Pharmacological agents and methods of treatment that inactivate pathogenic prokaryotic and eukaryotic cells and viruses by attacking highly conserved domains in structural metalloprotien and metalloenzyme targets

ABSTRACT

The invention relates to the treatment of viral, bacterial, parasitic, proliferative diseases, neurodegenerative diseases, inflammatory diseases, immunological diseases, transplanted organ rejection, and diseases produced by intoxication with heavy metals. The invention relates to the use of specific metal chelating agents including, furoic acid, 2-thiophenecarboxylic acid and their derivatives, analogs and structurally related chemicals as pharmacological agents that can be used effectively to disrupt and inactivate specific transition metal ion containing zinc finger structural motifs in metalloproteins and specific transition metal ion containing catalytic sites in metalloproteinases, which in turn, inactivate the pathogenic virus, pathogenic prokaryotic or eukaryotic cells which produces disease conditions. The preparations can be administered topically or for systemic use. The preparations are novel wide-spectrum antibiotics which have antiviral, antiproliferative, antineoplastic, antiangiogenic, antibacterial, antiparasitic, antiinfective, and anti-inflammatory effects and can be used in the treatment and prevention of diseases such as AIDS, cancers, untoward angiogenesis, pulmonary anthrax, malaria, inflammatory responses, Alzheimer&#39;s disease and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a division of application Ser. No. 10/161,981, filed Jun.4, 2002.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] None

BACKGROUND OF THE INVENTION

[0003] The invention relates to the treatment of viral, bacterial,parasitic, benign and malignant proliferative diseases,neurodegenerative diseases, inflammatory diseases, immunologicaldiseases, transplanted organ rejection diseases, and diseases producedby intoxication with heavy metals. The invention relates to the use ofspecific metal chelating agents including, furoic acid,thiophenecarboxylic acid and their derivatives, analogs and structurallyrelated chemicals as pharmacological agents that can be use effectivelyto disrupt and inactivate specific transition metal ion containing zincfinger structural motifs in metalloproteins and enzymatically activetransition metal ion containing sites in metalloproteinases, and othermetal containing motifs structural or functional, which in turn,inactivate the pathogenic virus, pathogenic prokaryotic or eukaryoticcells which produces disease conditions. The invention also includes theinactivation of any newly created biological pathogens and theirmetalloprotein products heretofore not recognized, such as those use inbioterrorism. Since radioactive and non-radioactive materials canintoxicate metalloenzyme systems involved in normal physiologicalfunctions, the agents of this invention are also intended to be used fordecontamination of animals and patients exposed to heavy metalsspontaneously or by the use in bioterrorism.

[0004] It will be appreciated that hereinafter the use of the followingterms:1)“metalloprotein disrupting agent” encompasses all of theintended functions of the invention and method including antiviral,antiinfective, antiinflammatory, anticancer, and so on; 0.2) the broadterm “antiinfective” is intended to include antiviral, antibacterial,antifingal, antiparasitic activities, as well as actions against anyother infective agent or organism whether natural or synthetic; 3) theterm “antiinflammatory” is intended to include any inflammatoryresponse; and 4) the term “decontamination of heavy metals” is intendedto include any use of these agents in the treatment of stable orradioactive heavy metal poisoning in man or animals. The term“biological response modifier” is intended to encompass any change inthe response of a prokaryotic or eukaryotic cell to a second agent afterinitial treatment with any of the agents of this invention. It will alsobe appreciated that the term “anti-inflammatory” is intended to includeall inflammatory responses of a metazoan organism such as production ofstress heat shock proteins, white blood cell infiltrates, swelling,pain, fever and so on. The term “analgesic” refers to a pain relieveragent that functions in inflammatory conditions.

[0005] The role of metal ion containing proteins in physiologicalactions and pathological responses including cancer, inflammation,proliferative diseases and infectious diseases have been intensivelystudy by many researchers. The inventors have studied the importantfunctions of proteins having amino acid sequences which specificallybind transition metal ions. For example, the inventors have determinedthe role of zinc finger proteins in cancer, proliferative diseases andviral diseases. Moreover, the inventors have determined the role ofnumerous metalloproteins, such as the role of iron-finger hormonereceptor proteins and zinc finger ribosomal proteins in carcinogenesisand aging.

[0006] A review of the literature reveals the critical role of metalcontaining proteins in physiological actions and pathological responsesincluding cancer, inflammation, proliferative, infectious diseases andheavy metal poisoning (Fernandez-Pol, JA, 2001). The inventors havestudied the important function of proteins having amino acid sequenceswhich bind metals, particularly transition metal ions. The inventorshave determined the important role metalloproteins in proliferative,inflammatory and infectious diseases. In additions, the inventors havedetermined the role of other metal ion containing protein complexes,such as the role of iron finger proteins in aging and carcinogenesis.

[0007] From the evidence reviewed, one can infer that development of avariety of drugs that control or neutralize metalloproteins may lead toa new therapeutic approach directed at controlling and preventing a widespectrum of viral diseases, bacterial diseases, fungal diseases, cancerand other diseases involving abnormal expression of metalloproteins.Furthermore, the results suggest that these agents may be useful toprevent transmission of viral diseases and prevent the progression ofother diseases. This review of the literature not only points out thelimits of our understanding of this system, but also indicates the needfor the development of new agents to control metalloproteins.

[0008] The specific features, objectives, and advantages of the instantinvention and its preferred embodiments will become apparent afterreview and comparison with the prior art as follows. Remarkably, theinstant invention provides agents heretofore not recognized, thatinteract with high specificity with structural metalloproteins andmetalloenzymes essential for viral and cellular functions. However, noneof these chelators are specific for zinc, in fact, some of them are morespecific for iron, and they may have chelated a variety of transitionmetals (29). Nevertheless, these studies indicate that zinc plays acomplex role in a dose and time-dependent manner in apoptosis.

[0009] The recent targeting of individuals and groups with the anthraxbacterium (Bacillus anthracis) spores is of great concern, particularlybecause the pulmonary form of this disease is most often fatal. Thisindicates the need to develop new antibiotics that will rapidly andeffectively destroy the B. anthracis which proliferates inside lung andlymph node macrophages. This invention contributes new wide-spectrumantibiotics suitable for the destruction of B. anthracis insidemacrophages which cannot perform their bactericidal functions in thepresence of such bacterium.

[0010] Although only a limited number of viruses are currently thoughtto be adequate for biowarfare, such as smallpox or Marburg virus, thereis a large number of viruses that can be made suitable and highlydangerous through genetic engineering manipulation or other selectionprocess. There are thousands of animal and human viruses that have thepotential to serve that purpose. Those viral agents that have not beenrecognized previous as credible biowarfare agents pose a present andclear danger, because there is no protection in the form of vaccines forprevention, no credible therapies, and no detection. The potential fordestructive pandemic consequences would be far greater than those of theanthrax bacterium and may be similar to that of ancient smallpoxpandemics. An opportunity exist to urgently correct and counteract thissituation by developing new effective, low toxicity wide-spectrumantiviral agents. This invention contributes new wide-spectrumanti-viral agents suitable for these purposes.

[0011] The next sections discuss the background of this invention withspecial reference to applications to abnormal physiology, infectiousdiseases, heavy metal toxicity, special applications to enzymes, andmetal complexes (Table 1).

Essential Viral and Cellular Zinc and Iron Containing Metalloproteins asTargets for Novel Antiviral and Anticancer Agents

[0012] In this section the inventors summarize the experimental data onthe role of a selected group of metalloproteins, particularly viral (v)and cellular (c) zinc finger proteins (ZFP) and iron containing proteinswhich are involved in cell proliferation, neovascularization, apoptosis,and viral infection. Furthermore, this review summarizes the dataembracing the hypothesis that disruption of certain metalloproteins bynovel pharmacological agents is a key factor in controlling viral andproliferative diseases. The primary goal of this review is to show thepotential therapeutic applications of ZFP disrupting agents, zincchelators and iron chelators in the control of viral diseases andcancer.

[0013] It is known that zinc or iron deficiency, resulting from exposureof culture cells to membrane-permeable Zn²⁺ or Fe²⁺-chelators, caninduced apoptosis in virally transformed cells while normal cells remainunaffected under these conditions. Apoptosis is possibly due tosimultaneous inactivation of vZFP, cZFP, and/or iron containingproteins, which are essential for maintenance of cellular and viralstructure and which are activated in virally transformed cells. Newinsights concerning apoptosis, viral metalloproteins, and novelantiviral agents will also be reviewed.

[0014] From the evidence reviewed, one can infer that development of avariety of drugs that control or neutralize vZFP may lead to a newtherapeutic approach directed at controlling and preventing a widespectrum of viral diseases and cancer. Furthermore, the results suggestthat these agents may be useful to prevent transmission of viraldiseases. Finally, these data not only points out the limits of ourunderstanding of these systems, but also directed the inventors to thecreation of the novel agents of the instant invention.

[0015] Due to the central importance and essential functions of viraland cellular zinc-finger proteins, the literature on these topics is nowrapidly expanding. Different aspects of ZFP functions, for example, inapoptosis induced by viruses, been reviewed in recent years. In thisarticle we will concentrate on the available information about variouszinc finger proteins of viruses, the drugs that inhibit their function,and cellular zinc finger proteins induced by virus infection in anattempt to critically evaluate some basic biological consequences ofmanipulating zinc finger proteins.

[0016] The background of the invention is presented as a brief summaryof information with some initial hypothesis supported by experimentalevidence at the molecular and clinical level. The inventors evaluate therole of a selected group of zinc finger proteins of cells and virusesinvolved in apoptosis and viral infection. Because of the importance ofribosomal proteins having zinc finger structures in viral replicationand cancer, those functions also are reviewed. Moreover, the authorsbriefly address the role of heat shock zinc finger proteins. Finally,the inventors summarize the data that leads to the known informationembracing the hypothesis that disruption of zinc finger proteins bynovel antiviral and anticancer agents is a key factor in controllingviral diseases and cancer.

[0017] Apoptosis.

[0018] Apoptosis, or single-cell death, is programmed cell death thatoccurs during normal homeostasis. Apoptosis is further defined bymorphological and biochemical characteristics which are regulated byevolutionary conserved genetic pathways common to metazoan organisms.Apoptosis serves a complementary but opposite role to mitosis in normalhomeostasis.

[0019] Apoptosis involves a complex network of pathways that interactwith each other to be either pro-apoptotic or anti-apoptotic.Developmental or environmental stimuli activate or inactivate specificgenes resulting in a series of biochemical degrading reactions thatresult in orderly cell disintegration. Apoptosis is induced by a varietyof events such as viral infection, anticancer agents, radiation, growthfactor deprivation, cytokines and hormones. Mutations of genes in thispathway eliminate the apoptotic response and have proven oncogenic. Forexample, over expressing the gene Bcl-2 or eliminating the gene p53alters the susceptibility to apoptosis and allows the proliferation ofdamaged cells which frequently results in cancer.

[0020] Zinc Chelating Agents and Apoptosis.

[0021] A recent review summarizes the evidence that apoptosis ismodulated by intracellular excess or deficiency of Zn²⁺ and presentssome mechanism by which Zn²⁺ may control apoptosis (Fernandez-Pol, etal, 2001). The major conclusions are: 1) zinc deficiency, resulting fromdietary deprivation or exposure of cultured cells to membrane-permeableZn²⁺ chelators induces apoptosis; 2) zinc supplementation with Zn²⁺ tothe media of cell cultures, can prevent apoptosis; and 3) anintracellular pool of chelatable Zn²⁺ plays a critical role inapoptosis, possibly by modulating the activity of endonucleases.

[0022] There is evidence that apoptosis is modulated by intracellularexcess or deficiency of Zn²⁺. Fragmentation of DNA and cytolysis areinhibited in certain systems when Zn2+ (0.8 mM) is added to the culturemedium. It is interesting to note that Ca2+/Mg2+-dependent endonucleaseactivity in isolated nuclei was inhibited when Zn2+ was added to themedium. These studies are consistent with the hypothesis that Zn2+prevents apoptosis by blocking the activation or inhibiting the activityof Ca2+/Mg2+-dependent endonuclease. Numerous reports have shown thatdepletion of intracellular Zn2+ by chelation can trigger apoptosis invirally transformed cells. For example, when leukemia cells were exposedto 1,10-phenanthroline, a Zn2+/Fe2+ chelator, DNA fragmentation and celldeath occurred, unless the chelator was neutralized by a transitionmetal ion added to the medium. Similarly, picolinic acid (PA) aZn2+/Fe2+ chelator, induces apoptosis in many cells, including leukemiacells by chelating a pool of intracellular Zn2+/Fe2+, since influx ofZn2+/Fe2+ prevented apoptosis in the presence of PA, while chelation ofZn2+/Fe2+ induced apoptosis.

[0023] Because Zn2+ plays a role in many cellular functions, and becauseit is an structural component of zinc finger proteins which areessential in cell replication, there are many sites in the apoptoticpathway that can be potentially modulate by zinc and zinc chelators. Anumber of investigators have shown that apoptosis can be induced if theintracellular level of Zn2+ are reduced using chelators. For example,N,N,N′,N′-tetrakis-2-pyridyl methyl-ethylene diamine (TPEN) added tocultured cells induces apoptosis. These experiments add additionalsupport to the hypothesis that changes in intra- and extracellular zinccan modulate apoptosis. However, none of these chelators are specificfor zinc, in fact, some of them are more specific for iron, and they mayhave chelated a variety of transition metals. Nevertheless, thesestudies indicate that zinc plays a complex role in a dose andtime-dependent manner in apoptosis.

[0024] Viruses relevant to human disease such as Smallpox, Ebola virus,Marburg virus, Lassa virus, Papillomavirus, Herpesvirus, andRetroviruses, including the AIDS virus, are all capable of inducingapoptosis. Viruses encode genes that both stimulate and suppressapoptotic cell death. These viral proteins interact with cellularpro-apoptotic (death factors) and anti-apoptotic (survival factors).Viral (v) and cellular (c) Zinc finger proteins (ZFP) are involved inapoptotic cell death. A pool of chelatable intracellular Zn²⁺ plays acritical role in viral and cellular apoptosis, possibly by modulatingZFP structure. In virally transformed cells apoptosis can be induced byintracellular deficiency of Zn²⁺ while normal non-infected cells remainunaffected.

[0025] Since 1980, Fernandez-Pol et al are studying the modulation ofboth v-ZFP and c-ZFP by a class of novel Zn²⁺/Fe2+ chelating,broad-spectrum antiviral agents which may form ternary complexes withthe zinc atoms contained in ZFP (42-60). In numerous experiments, wefound that these wide-spectrum antiviral agents block viral replicationand induced apoptosis in virally transformed cells in culture. Theseagents also interfere with abnormally expressed c-ZFP produced byspontaneously or radiation transformed cells in culture. Thus, thesestudies provide evidence for a close correlation between interferencewith ZFP of both viral and cellular origins and apoptosis in transformedbut not in normal cells.

[0026] Iron and Zinc Finger Proteins.

[0027] Transition metal ions at physiological concentrations, such aschromium, zinc, iron, cobalt, and copper, are essential elements forbiological functions; however in higher quantities they are toxic(Fernandez-Pol, et al, 2001). Evidence indicates that elevated levels ofiron contribute to carcinogenesis. Two main factors are important iniron induced oncogenesis: 1) The capacity of iron to generate highlyreactive free radicals which damage DNA; and 2) the increase ironrequirement by rapidly proliferating transformed cells, which isrequired for DNA replication (ribonucleotide reductase) and energyproduction (within the mitochondrial in key enzymes of the redox systemsof the respiratory chain). Studies with iron chelating agents such aspicolinic acid and desferoxamine have contributed significantly to theunderstanding of differential mechanisms of growth regulation in normaland transformed cells (Fernandez-Pol et al, 2001). It is known that ironinduces mutagenesis and/or carcinogenesis, but the detail mechanism ofiron-induced oncogenesis is unknown.

[0028] Initial in vitro studies have demonstrated the ability of cobaltand cadmium to structurally reconstitute the zinc finger domains in anactive form. In contrast, nickel and copper bind to zinc fingerproteins, but are unable to restore the DNA binding capacity. Thesestudies suggest that heavy metal incorporation into zinc finger may beimportant in metal-induced toxicity. Recently, it has been found that aniron-substituted zinc finger may generate free radicals which damage DNAand potentially induced carcinogenesis. The estrogen receptor (ER) is aligand-activated transcription factor whose DNA-binding domain (ERDBD)is of the type Cys4-Cys4, which coordinate two zinc atoms, forming twozinc finger domains. The capability of iron to replace zinc in zincfinger, denoted the iron finger, was demonstrated in a series ofexperiments both in vivo and in vitro. Iron has the ability tosubstitute for zinc in the ERDBD as demonstrated by mobility shift andmethylation interference assays of iron finger, which show specificrecognition of the estrogen response element. The DNA binding constantsfor both in vivo and in vitro iron-replaced zinc fingers were similar tothat of the native zinc-containing finger. Atomic absorption analysisshowed a ratio of 2:1 iron atoms/mol of ERDBD protein. Remarkably, theiron finger in the presence of hydrogen peroxide and ascorbate generateshighly reactive free radicals (hydroxyl), producing a reproduciblecleavage pattern to the DNA of the estrogen response element. The closeproximity of the zinc finger to DNA, as found in the computer modeledstructure, suggests that the iron-substituted zinc finger may generatefree radicals while bound to genetic regulatory response elements,leading to degradation of DNA and/or carcinogenesis.

[0029] Zinc Finger Ribosomal Proteins

[0030] Fernandez-Pol et al have shown that human metallopanstimulin(MPS-1)/S27 ribosomal protein is a ubiquitous 9.4-kDa multifunctional“zinc finger” protein which is expressed at high levels in a widevariety of cultured proliferating cells and tumor tissues. The humanMPS-1 gene and its relationship to human cancer cell growth wasdiscovered by Fernandez-Pol et al in 1989, using human MDA-MB468 breastcancer cells stimulated with specific growth factors and serum. Sincethat time, research has consistently demonstrated that both MPS-1 mRNAand protein are involved in cancer cell growth as demonstrated byincreased levels of MPS-1 mRNA and protein found in numerouspathological tissue specimens obtained from various types of humancancers, such as prostate, breast, lung, colon, endometrium, uterinecervix, vulva, and melanoma. These results indicate that the MPS-1antigen is a ubiquitous tumor marker which may be useful in detectionand prognosis of various types of malignant neoplastic conditions. Theresults of other experiments indicate that MPS-1 is involved in proteinsynthesis, repair of damaged DNA, digestion of mutated mRNA,anti-apoptosis and rapid cell proliferation. Thus, the informationavailable indicate that MPS-1 is a multifunctional S27 ribosomal proteinrelevant to numerous oncogenic processes which can be used as aubiquitous tumor marker in various clinical assays. More recently,MPS-1/S27 ribosomal protein has been shown to be increased in virusinfected cells, in parasites such as Toxoplasmosis and Malaria, in yeastproliferative capacity, and in macrophage activation in human melanomas(NCBI, National Cancer Institute Data Bank; Fernandez-Pol, 2001).

[0031] It is important to note at this point that there are many reportsindicating a connection between overexpression of some genes encodingribosomal proteins and cancer. There is evidence that a number of otherribosomal proteins have additional functions separated from both theribosome and protein synthesis. Zinc finger motifs are characteristicsof numerous ribosomal proteins, allowing them to bind to nucleic acids.This binding ability offers a potential mechanism for ribosomal proteinsto interfere in both transcriptional and translational mechanisms. Forexample, the rat ribosomal protein S3a is identical to the product ofthe rat Fte-1 gene which encodes the v-fos transformation effector. S3ais involved in the initiation of protein synthesis and is also relatedto proteins involved in the regulation of growth and the cell cycle. Ratribosomal protein L10 is homologous to the Jun-binding protein and to aputative Wilm's tumor suppressor. Taken together, the findings ofribosomal proteins with oncogenic, tumor supressor, or cell cyclefunctions, indicates extraribosomal functions of certain ribosomalproteins related to oncogenesis.

[0032] Zinc Finger Heat Shock Proteins and Viral Activation

[0033] The involvement of zinc fingers in protein-protein interactionsextends beyond the control of gene expression. In numerous proteins thezinc finger domains have been implicated in mediating homodimerizationor heterodimerization (Fernandez-Pol et al, 2001). Prokaryotes andeukaryotes express numerous heat shock proteins (Hsps) in response tostress, including heat shock, exposure to heavy metals, hormones andviral infections.

[0034] The stress response which include numerous forms of physiologicaland pathological stress is involved in viral infection. A prominentfeature of this response is the synthesis of a discrete set of zincfinger proteins, known as the heat shock proteins, which at present aredenoted molecular chaperons. During infection by certain viruses, heatshock proteins act as intracellular detectors that recognize malfoldedproteins. Researchers have found that certain DNA viruses are able toactivate heat shock proteins. For example, the Hsp70 (DnaK) is inducedby adenovirus, herpes virus, cytomegalovirus, and other viruses.

[0035] One of the most interesting proteins involved in the viralinfection response is the DnaJ, a heat shock protein which functions inthe control of protein folding within the cell. DnaJ proteins containtwo CCCC zinc finger motifs, defined by the J domain, which is essentialfor stimulation of the Hsp70 ATPase activity. Thus, the results indicatethat there is a relationship between the stress response and thecytopathic effects of certain viruses such as herpes viruses,poxviruses, and hepatitis C viruses.

[0036] The response of cells to stress, such as exposure to UVradiation, chemicals, bacteria, parasites, fungus or viruses is alsoassociated with the induction of heat shock proteins. Hsp70 has aprotective role in inflammation, infection, and regulatory roles incytokine biosynthesis. Hsp70 exists in the cells in equilibrium betweenits free state, in the cytoplasm, and its bound state, protectingproteins in the nucleolus, interacting with ribosomal proteins to eitherrefold some of the unfolded ribosomal proteins or by solubilizing thedenatured ribosomal proteins to facilitate their use and increase theturnover rate. During release as a result of the heat shock, and as thenucleolus begins to recover its normal activities, a significantproportion of Hsp70 returns to the cytoplasm. This protein-proteininteraction may have important implications for viral replication.

[0037] Thus, cellular inflammatory responses to viral infection are partof the organism defense against viruses. Zinc finger proteins,therefore, may be a key to the control of the cellular inflammatoryresponse. Agents which can modify the zinc finger heat shock proteinsmay be useful in controlling the stress response.

[0038] Viral Zinc Finger Proteins are Highly Conserved Structures.

[0039] All viruses depend on their ability to infect cells and inducethem to make more virus particles. If the virus is successful the cellsalmost invariably die in the process, and that process have been shownto be apoptosis in numerous instances. Other viruses can integrate itsDNA in the cellular DNA and remain inactive for long periods. Thenucleic acid genome of viruses is always surrounded by a protein shell,denoted capsid, which is composed of nucleocapsid proteins, and someviruses also have a lipid bilayer membrane, termed an envelope, whichenclose the nucleocapsid proteins.

[0040] Viral ZFPs have been identified in at least two thirds of allviruses studied (Fernandez-Pol et al, 2001). Examples of families ofviruses using metalloproteins such as ZFP, zinc ring proteins ortransition metal ion-dependent enzymes for replication, packaging andvirulence are Arenaviridae, Reoviridae, Rotaviridae, Retroviridae,Papillomavirinae, Influenza, Adenoviridae, Flaviviridae (Hepatitis C),Herpesviridae, and Orthomyxoviridae (Influenza viruses). Viral ZFP arestructural virion proteins essential for viral replication and packagingof the virus inside infected cells. Deletion of zinc finger domains inspecific vZFP is lethal to the virus. Since the zinc finger domains ofvZFP are essential for viral survival functions, they are conservedthroughout evolution and there are no known mutants of the vZFPdomain(s). Because the viral zinc finger domain(s) representindispensable site (s) on the vZFP that can be attacked by one ormultiple drugs, vZFP are ideal and primary drug targets for the nextgeneration of antiviral agents (Fernandez-Pol et al. 2001).

[0041] A computer search of all known viruses reveals highly conservedstructures in their nucleocapsid (NC) proteins and other essential viralproteins. All viral NC proteins contain sequences of about 20 aminoacids with 4 invariant residues, CCHC or other combinations, whichchelate zinc through histidine imidazole and cysteinic thiolates with aKd les than 10⁻¹³. These structures are denoted viral zinc fingers, andare highly conserved in numerous families of viruses. Examples ofviruses which posses zinc finger NC proteins and other zinc bindingproteins are show in Table I. These metal binding proteins are highlyconserved in nature, and they perform essential functions in viralinfectivity. It has been shown that mutations of the chelating residuesin the zinc fingers produces a non-infectious virus. Furthermore,chelating agents have been shown to inactivate viruses. Thus, disruptionof these proteins by specific agents results in viral inactivation. Ithas been suggested that the course of numerous viral diseases, such asgenital warts, genital herpes, smallpox, chickenpox, influenza, viralhepatitis, etc, can be altered by inhibiting essential viralmetalloproteins utilized during the viral infection cycle (Fernandez-Polet al, 2001).

[0042] Papilloma virus infection results in a number of proliferativediseases in humans including warts induced by type 4 human papillomavirus (common warts). Moreover, papilloma virus can cause plantar ulcersas well as plantar warts. Human papilloma virus infection of the uterinecervix is the most common of all sexually transmitted diseases. Commonlyknow as genital warts, this wide spread virus infection is a seriousdisease that potentially can develop into cervical cancer. Since thevirus is permanently present in cells, infection recurs in a significantpercentage of patients.

[0043]Condylomata acuminata, also denoted genital warts, are benignepithelial growths that occur in the genital and perianal areas andcaused by a number of human papilloma viruses (HPV) including types 6,11and 54. These are low risk viruses which rarely progress to malignancy.However, high risk viruses such as HPV-16 and HPV-18 are associated withcervical intraepithelial cancer.

[0044] The actions of HPV are mediated by specific viral-encodedproteins which interact and/or modulate cellular DNA and proteins toproduce abnormal growth and differentiation of cells. Two proteins ofthe HPV viral genome, E6 and E7, are well conserved among anogenitalHPV's and both contribute to the uncontrolled proliferation of basalcells characteristics of the lesions. The E7 oncoprotein is amulti-functional protein with transcriptional modulatory and cellulartransforming properties. The E7 oncoprotein is a zinc finger protein.

[0045] A strong correlation between zinc binding and transactivationactivity of E7 has been documented. The HPV-16 E6 protein is a zincfinger protein that binds DNA and has transcriptional activity whichdepends on the formation of the zinc fingers. E6 protein can complexwith the cellular tumor suppressor protein p53 and it is necessary withE7 protein for the immortalization of primary human epithelial squamouscells. Only two proteins of HPV are consistently expressed andintegrated in keratinocytes, the E6 and E7 zinc finger proteins. The E6and E7 proteins are responsible for continuous cell proliferation. Abouttwenty HPVs are associated with ano-genital lesions and all transformedkeratinocytes of these lesions contain E6 and E7 zinc finger proteins.The E6 and E7 regulate growth and transformation by interfering withcellular p53 and pRb proteins, respectively. Thus, one should be able tocontrol HPV by inactivating E6 and E7, the critical zinc finger proteinswhich are required for replication. When replication of the virus ishalted, apoptosis of the virally-infected cells must occur. Thus, onecan alter the epidemiology of, for example, carcinoma of the uterinecervix by interfering with the functions of zinc finger proteins.

[0046] Herpes viruses are highly disseminated in nature. Herpes virusesvary greatly in their biological properties and the clinicalmanifestations of diseases they cause. In humans eight herpes viruseshave been isolated to date: 1) herpes simplex virus 1 (HSV-1), herpessimplex virus 2 (HSV-2), cytomegalovirus (HCMV), varicella-zoster virus(VZV), Epstein-Barr virus (EBV), human herpesvirus 6 and 7 (HHSV6 andHHSV-7). More recently the existence of HHV8 as a causative agent ofKaposi sarcoma has been documented. The known herpesviruses share twosignificant biological properties relevant to this invention: 1) allherpesviruses specify a large array of enzymes involved in nucleic acidmetabolism, including ribonucleotide reductase, an iron containingenzyme; and 2) they possess major zinc finger DNA-binding proteinsrequired for DNA replication.

[0047] Retrovirus virions contain a diploid genome consisting of an RNAcomplex formed by the association of two identical unspliced viral RNAmolecules. In mature virions, RNA molecules are tightly bound to viralzinc finger proteins, denoted nucleocapsid proteins (Ncps). RetroviralNcp is produced after the gag gene product (Pr55gag), has been processedby the viral protease. The Ncps are highly conserved in all knownretroviruses. Point mutation of the cysteine and histidine residues ofthe zinc finger domain of NCp7 results in a radical reduction of genomicRNA packaging, and this results in a drastic decrease in viralinfectivity. Further studies indicate that NCp7 plays a key role inseveral other steps of the viral life cycle.

[0048] The human immunodeficiency virus (HIV) encodes several regulatoryproteins that are not present in other retroviruses. The nucleocapsid p7protein of HIV has been targeted by the inventors and otherinvestigators for treatment of HIV viral infections. The p7 protein isrequired for the correct assembly of viral RNA in newly formed virusparticles. The p7 protein contains two zinc fingers that are criticalfor the recognition and packaging of the viral RNA. Thus, agents thateffectively attack the two zinc finger domains of the HIV virusnucleocapsid p7 protein inside infected cells will decrease the overallnumber of viral infective particles.

[0049] The influenza virus is not integrated with DNA and thus may bevulnerable to attack by the specific antiviral agents of this invention.The influenza viruses are dependent upon viral Zn²⁺ metalloproteases forspecific viral functions. Processing of critical proteins of influenzavirus is mediated by virus-encoded Zn²⁺ metalloproteases. It is ofinterest for this invention that the most abundant virion protein and atype specific antigen of influenza viruses, the M1 protein, is a zincfinger protein. Furthermore, this protein is involved in packaging ofthe influenza virus. Thus, inhibition of influenza virus Zn²⁺metalloproteinases and/or zinc finger protein M1 by the agents of thisinvention presents an opportunity for controlling the progression ofinfluenza virus infection.

[0050] The poxviridae is a large family of complex DNA viruses thatreplicate in the cytoplasm of vertebrate and invertebrate cells. Themost notorious virus of this family is the variola virus that causessmallpox. Infectious poxvirus particles contain a complex transcriptionsystem. A large number of virus-encoded enzymes and factors are packagedin the virus particle. For example, RNA polymerase, a zinc requiringenzyme, is involved in early transcription. Furthermore, both the smallcatalytic subunit and the large regulatory subunit of ribonucleotidereductase are virus-encoded proteins and closely resemble theireukaryotic counterparts both structurally (80% homology) andfunctionally. The synthesis of ribonucleotide reductase, is inducedrapidly after vaccinia virus infection. Catalytic activity of the smallsubunit is inhibited by hydroxyurea. Furthermore, some of the earlyviral and cellular transcription factors utilized by the smallpox virusare zinc finger proteins.

[0051] Filoviruses, which cause deadly haemorragic fevers, are a largegroup of viruses that have non-segmented negative-strand (NNS)RNA astheir genomes. The two main types are the Marburg and the Ebola virus.The nucleoproteins of these viruses interact with the linear RNA genomeand also with cellular and ribosomal zinc finger proteins to performspecific viral functions. Thus, filoviruses are susceptible toinhibition by the agents of this invention.

[0052] There are numerous examples of families of viruses which utilizezinc finger proteins, zinc ring proteins and/or transition metalion-dependent enzymes for specific viral functions. These viral proteinsplay an essential role in the structure, replication and/or virulence ofviruses such as Reoviruses, Rotaviruses, Hepatitis C viruses as well asnumerous other viruses.

Zinc Finger Proteins: Targets for Pharmacological Therapy

[0053] Background.

[0054] The National Cancer Institute has identified ZIP as the nexttarget for antiviral drugs (USA Federal Register, 60, No. 154, 1995).Several laboratories are evaluating new antiviral drugs targeted tomodify ZFP. These products are targeted towards modification of theamino acid cysteine, which is the binding site for zinc in zinc fingerproteins. This section focuses on developments in the antiviral fieldrelated to inhibititors of zinc finger proteins of HIV, HPV, and HSV.

[0055] It has been known for many years that the structural andbiological properties of viruses can be altered by chelating agents. Forexample, treatment of rotaviruses with chelating agents such as EDTA (10mM) results in a single-shelled, double-layered, non-infectious viralparticles. Moreover, in vitro exposure of various retroviruses to thechelating agents such as EDTA or EGTA in millimolar concentrationsresults in partial disintegration of viral membranes. Thus,disintegration and degradation of retroviruses and rotaviruses can beaccomplished by chelating agents.

[0056] There are several chelating agents that eject the coordinatelybound zinc atom from HIV zinc finger proteins. For example, Otzuka et alreported that novel zinc chelators inhibit the DNA-binding activity ofzinc finger proteins of HIV. In addition, The Tat trans-activator, is asmall protein of 75-130 amino acids, which may form a zinc-fingerdomain. Since HIV-1 lacking Tat replicates poorly and does not causecytopathic effects, approaches to interfere with Tat may be useful intreating AIDS. The cysteine-rich domain of Tat binds divalent cations,either two Cd2+ or two Zn2+ atoms. Whether the cysteine-rich residuesform a Zn2+ finger or a lattice binding pockets for divalent cations isunknown. The pol gene also has a zinc finger amino acid sequencesuggesting that chelation chemotherapy may have a role in the treatmentof AIDS.

[0057] At least three efficient approaches may be used to design novelclasses of inhibitors of viral ZFP activity that directly attackvZFP: 1) disruption of the zinc finger domain by modification of thecysteine residues which are the binding sites for Zn²⁺ in the vZFP,resulting in the ejection of zinc ion; 2) removal of the zinc from thezinc finger moiety by specific chelating agents, which results ininactivation of the vZFP; and 3) specific chelating agents that form aternary complex at the site of zinc binding on vZFP, resulting ininhibition of the DNA or RNA binding activity of vZFP. Since theseantiviral agents attack highly conserved structures in the virus theymay circumvent the emergence of drug resistant mutants. Furthermore, thebasic mechanisms of action of the novel antivirals (1 through 3, above)may be enhanced in viral disease if the antiviral agents which directlyattack metalloproteins of the virus simultaneously attack cellularmetalloproteins implicated in the pathogenesis of viral disease. Hence,the novel antivirals may also prove to be effective against cellularzinc finger-containing proteins such as ribosomal ZFP and heat shockproteins which are involved in viral infection. These cellular proteinsare induced by the virus for specific viral functions such asreplication, propagation, or as an inflammatory response of the cells tothe virus.

[0058] The specificity of these agents may be due to cellularspecificity, in which virally infected cells express cellular and viralZFPs that are not expressed by normal uninfected cells in their basal orproliferative state. Another primary mode of action of these agentscould be receptor specificity, in which vZFP act as receptors forspecific zinc ejecting agents, or specific chelating agents which bindto vZFP and form an inactive ternary complex consisting ofvZFP-Zn-chelating agent. Thus, vZFP may act as receptors for new agentsthat can form ternary complexes with vZFP.

[0059] Disruption Of Viral Zinc Fingers Of HIV By Chemical Agents

[0060] The HIV virus represents a daunting challenge for chemotherapy. Amajor problem with the treatment of HIV-1 infection is the emergence ofdrug resistance. HIV-1 nucleocapsid protein zinc fingers are targets ofchoice because they are mutationally intolerant and are required forboth acute infection and virion assembly. After treatment with nontoxicdisulfide-substituted benzamides which attack zinc fingers, thefollowing has been observed with HIV virus: 1) inactivation of cell-freevirions; 2) inhibition of acute and chronic viral infections; and 3)broad anti-retroviral activity. More important, resistant mutants havenot been detected.

[0061] In all retroviruses, except spumaretroviruses, the nucleocapsid(NC) proteins contain one or two copies of a conserved zinc fingerdomain. This motif, represented as C—X₂—C—X₄—H—X₄—C (C, cysteine; H,histidine; X, other amino acids), thereafter denoted a CCHC box orretroviral zinc finger, is different in different retroviruses, andcoordinates a zinc ion in the virion. The histidine imidazole andcysteine thiolates chelate zinc (Kd<10⁻¹³). The CCHC box is one of themost highly conserved motifs in retroviruses. All mutations in the zincfinger domain which have been described to date have been lethal for thevirus. The HIV-1 NC protein contains two zinc fingers separated by just7 amino acids. The zinc fingers of NC p7 protein are required for bothearly events in virus infection and for packaging genomic RNA.

[0062] Rice et al proposed that the HIV-1 NC protein is an ideal targetfor drug development because of the following reasons: 1) the two zincfingers are indispensable sites on the same protein that can be attackby a single drug; 2) Simultaneous mutations in each zinc finger would berequired for the development of drug resistance; and 3) the closeproximity of the targets greatly reduces the probability ofrecombinatorial events.

[0063] Numerous compounds that fracture the CCHC zinc finger alsoinactivate the HIV-1 virus. These products have been suggested to beeffective for a broad range of human and animal diseases, includingdrugs to treat cancer and viral infections. The zinc fingers ofretroviral NC proteins are prime antiviral targets because of theconservation of the Cys and His chelating residues and the absoluterequirement of these fingers in both early and late phases of retroviralreplication. Thus, drugs that interact with the CCHC zinc fingerstructures of HIV-1 and other retroviral nucleocapsid proteins may beextremely useful to halt the propagation of the virus.

[0064] A core HIV viral protein, denoted p7 nucleocapsid (NC), has twozinc fingers. The zinc fingers are involved in binding and packagingviral RNA into new virions which bud off from the infected cells.Experiments have demonstrated that when the two zinc fingers aredeleted, the new budding virions are unable to incorporate RNA,resulting in non-infectious viral particles. Furthermore, the p7 NCprotein participate in the process of reverse transcription. The exactrole of p7 in this process is not clear, but it is thought that the p7NC protein binds to the RNA molecule while the reverse transcriptaseenzyme generates DNA from the RNA template. More important, HIV p7 NP inwhich the zinc fingers have been deleted is unable to infect susceptiblecells. The zinc fingers are therefore essential for both phases of theviral life cycle: packaging and transcription.

[0065] A number of drugs that interact with zinc fingers work byejecting Zn from the zinc finger, resulting in an afunctional protein.The same drugs were active against HIV in tissue culture. One of thedrugs is disulfiram (Antabuse), a drug that is frequently used for thetreatment of alcoholism. However, this drug was ineffective in monkeyscarrying SIV. Rice et al reported that C-nitroso compounds inactivateHIV-1 and eject zinc from the virus. Retroviral zinc-fingers of the typeCCHC bind zinc stoichiometrically and with high affinity (dissociationconstant Kd, about 10⁻¹² M). Under physiological conditions, a 10-foldexcess of EDTA removes only 50% of the zinc from the N-terminal CCHCzinc finger domain of the HIV-1 nucleocapsid protein, indicating thatthe chelating agent EDTA is inefficient in removing Zn from ZFP. Todetermine whether 3-nitrosobenzamide (NOBA) is capable of efficientlyejecting Zn from retroviral-type zinc-fingers, Rice et al used NMR tostudy an 18-residue peptide with sequence corresponding to theN-terminal zinc-finger domain of the HIV-1 nucleocapsid protein, denotedZn (HIV-1-F1). The ¹H-NMR spectrum showed that addition of NOBA resultsin the loss of signals generated by the zinc-bound histidine, and theappearance of zinc-free histidine signals.

[0066] Rice et al have also proposed that disulfide benzamides possess awide-spectrum antiretroviral activity in cell culture by “attacking” thetwo zinc fingers of the HIV-1 nucleocapsid protein (NCp7) and ejectingthe zinc. Nucleocapsid is present in the core of all retroviruses. NCp7binds to the dimeric viral RNA genome. Mutagenesis of any of thecysteines of histidines in the ZF of HIV-1 NCp7 generates virions withdefective RNA encapsidation and noninfectious particles. Theseobservations indicate that compounds which specifically destroy thecoordination of the Zinc to the NCp7 will have an antiviral effect.Further experiments demonstrated that the antiretroviral activity ofthese compounds is due to inactivation of NCp7.

[0067] Finally, one of the great problems with antiretroviral agentscurrently in use is the ability of the virus to generate mutants whichare resistant to the therapeutic agent and which are able to replicatewith the same efficiency as the wild-type. This problem could be solvedif the target protein has no alternative structures capable of replacingthe original target protein. The properties of the zinc fingers innumerous viral proteins indicate the they are the proper targets toavoid mutation: The zinc finger is absolutely conserved in retroviruses(except spumaretroviruses), and all mutants in the zinc finger aredefective with respect to infectivity. However, it is unclear whethermutants defective in zinc fingers may arise in certain circumstances.

[0068] Inhibition of Viral and Cellular Ribonucleotide Reductases (RR)by Iron Chelating Agents: Implications for Therapy of HSV and HIV.

[0069] Antivirals for the treatment of herpes infections such asacyclovir, ganciclovir and foscarnet have had a significant impact onthe management of herpesvirus infections. However, the use of theseagents has resulted in an increase emergence of drug-resistant virusstrains. The need for new classes of anti-HSV compounds with novelmechanisms of viral inhibition is becoming increasingly apparent asmutants resistant to conventional antiviral agents emerge.

[0070] When a virus infects a cell, it could induce its host to makedoxyribonucleotides for viral DNA replication by means of the cellularenzyme ribonucleotide reductase, or the virus, as in the case of HSVcould carry its own specific RR genes which are expressed in the hostcells and produce a new enzyme.

[0071] Iron chelators inactivate the RR of HSV. Since iron restores theactivity of RR, the chelators inactivate the RR by directly removing itscatalytically essential iron. Interestingly, there are certain chelatorssuch as (348U87)2Fe and (A1110U)2Fe that also inactivate the viral RR.It is conceivable that the antiviral-Fe-RR forms a ternary complex thatprevents the catalytic function of the Fe2+, labilazing the enzyme-boundiron to dissociation.

[0072] Numerous herpes viruses, such as herpes simplex (HIV-1 andHIV-2), Epstein-Barr virus (EBV), varicella-zoster virus (VZV),pseudorabies virus (PRV), and equine herpesvirus type I (EHV-1), andnumerous other herpes viruses encode cellular ribonucleotide reductase(RR) activities. RR, which is formed by the association of twononidentical subunits (R1 and R2), catalyzes the reduction ofribonucleoside diphosphates to their 2′-deoxy derivatives which are keyintermediates in DNA biosynthesis. There is increasing evidencesupporting the essentiality of RR in viral replication. Numerousorganisms, including herpes viruses, bacteria, and mammals, encoderibonucleotide reductases the share a number of common characteristics.Two important characteristics of RR are the presence of a stable tyrosylfree radical and the dependency of Fe (III) for catalytic activity. Thesmaller (R2) subunit contains the iron and tyrosyl radical and thelarger (R1) contains thiols which are redox active and provide thehydrogen for nucleotide reduction. The association of R1 and R2 arerequired for catalytic activity. Thus, a potential approach forantiviral therapy would be the utilization of peptides that can inhibitenzymatic activity by preventing the association of R1 and R2 subunits.However, since iron is required for catalytic activity, a potential,less specific, strategy for antiviral therapy are iron chelating agents,which would deplete iron from the cells, and may have a significantactivity against herpes viruses. In 1998 picolinic acid was tested at 3to 1.5 mM on cultured Human Foreskin (HF) cells infected withHSV-2-strain G and it was found to cause apoptosis of HF infected cells.The specificity of the iron chelators may be cellular specificity ratherthan viral specificity: infected cells enter apoptosis versusnon-infected cells which remain unaffected.

[0073] It is relevant to mention that cellular RR is not only animportant virulence factor for herpes viruses, but that cellular RR isalso involved in the virulence of HIV. It has been suggested that theinhibition of RR with agents such as hydroxyurea could have a possibleapplication in the treatment of AIDS. Giacca et al have foundsynergistic antiviral actions of ribonucleotide reductase inhibitors and3′-azido-3′-deoxythymidine on HIV-1. RR inhibitors reduce the cellularsupply of DNA precursors (dNTP) by interfering with their de novosynthesis. A secondary effect is the stimulation of the uptake anphosphorylation of extracellular deoxynucleosides, including theiranalogs such as 3′-azidothymidine (AZT). Both effects are important toHIV replication, which requires dNTP and is impaired by the triphosphateof AZT. A clear synergism between AZT and RR inhibitors was observed atnontoxic doses.

Molecular Biology of Carcinogenesis and Aging

[0074] The cancer phenotype consists of several distinct characteristicssuch as indefinite proliferative life span, anchorage-independentgrowth, low growth factor requirements, neovascularization, invasion andmetastasis. A common characteristic of tumor cells is the constantoverexpression of glycolytic and glutaminolytic enzymes, which resultsin altered carbohydrate metabolism. In addition, cancerous cells cansynthesize their own growth factors, which leads to cell proliferationthat is independent of the otherwise carefully regulated supply ofgrowth factors and growth-related hormones. Moreover, growth factors areinstrumental in the invasive characteristics of cancer cells. Forexample, Vascular Endothelial Growth Factor (VEGF) activates theproliferation of endothelial cells which results in the creation of newblood vessels. Most interesting, growth factors can also activate matrixmetalloproteinases (MMPs) which are able to degrade the extracellularmatrix. Remarkably, one of the prominent features of MMPs is that manyof these genes are inducible by growth factors, cytokines, carcinogenicagents (e.g. phorbol esters), chemotherapeutic agents (actin stressfiber-disrupting drugs), radiation, and oncogenic cellulartransformation. MMPs gene expression may also be down-regulated bytransforming growth factors, retinoic acids and glucocorticoids. Thus,MMPs are fundamental enzymes in both the invasive process and metastaticdisease and are susceptible to pharmacological control. The developmentof potent synthetic inhibitors of MMPs had led to clinical trials totreat patients with cancer.

[0075] There are growth factors and oncogenes produced by viruses whichillustrate the complexity of the growth regulatory mechanism and theoncogenic process in vivo. For example, the complex smallpox virus whichhas recently acquire new notoriety due to its potential use inbioterrorism, contains a gene that encodes soluble EGF protein whichpromotes cell proliferation and is detected in all poxviruses genera.Furthermore, the family of poxviruses produce interleukin-1 betareceptor which blocks IL-1 beta cellular defense activity. TumorNecrosis Factor (TNF) is also produced by poxviruses and contributes tovirulence in the form of apoptosis. All these poxviruses virokines andviroreceptors gene products contribute to the pathogenesis in the formof production of granulomatous proliferative lesions, and benign tumorsas in the case of myxomatosis of rabbits. It has also been reported thatthe tat protein of the human immunodeficiency virus (HIV), a viralregulatory gene product, possesses growth stimulatory activity incertain cell types. Oncogenic proteins of human papilloma viruses suchas the E6 and E7 zinc finger proteins are also able to immortalizenormal cells in vivo and in vitro. These oncogenic proteins are involvedin human cancers such as the carcinoma of the uterine cervix.

[0076] The life spans of animals are genetically controlled and new dataexist to support common mechanisms to control the number of times a cellwill divide before it can no longer divide (senesce). A fundamentalcharacteristic of normal cells is their limited ability to proliferatein culture. Invariably, after an initial mitotic period in culture,normal cells from humans and most other species suffer a gradual declinein their ability to proliferate. Eventually, the decline becomesirreversible. This progression towards a lower activity state has beentermed “cellular senescence”. Cellular senescence has been studied mostoften in cultures of human fibroblasts (e.g. WI-38 cells). Numerousstudies have indicated that cellular senescence in culture reflectsaging in vivo. More recent studies have suggested that senescentfibroblasts are unable to proliferate, at least in part, because ofselective repression of genes involved in transcriptional activity, suchas a protooncogene designated as c-fos.

[0077] Cancer in humans and animals results from a multistep processwhich is described in experimental model systems as initiation,promotion, conversion, and progression. Each step in the processrepresents the selection of cells that have acquired the ability tosurmount extra and intracellular growth regulatory signals. Thecytogenetic evidence of multiple chromosome abnormalities in most tumorcells and the progressive aberrant chromosome structures that can beobserved during tumor progression are also evidence for multistepprocess. Since the tumorigenic process is of rare occurrence, multiplelevels of control must be operative to prevent the emergence of suchcells in metazoan organisms.

[0078] The common cancers of the adult, including colon, lung, prostateand breast develop by stepwise accumulation of mutations affecting bothoncogenes and tumor suppressor genes. These mutations accumulategradually over time and extensive genetic changes are necessary toproduce a highly malignant cell. For example, benign adenoma of thecolon usually have a single gene change. As they progress towardmalignancy they acquire 3 or 4 more gene alterations. These multiplechanges may occur in a specific order. However, it is very likely thatthe number of changes rather than a precise sequence is important forcancer development. Thus, the non-specific cellular changes as the cellages continue to contribute to shift the balance of control from tumorsuppressor genes to oncogenes.

[0079] There are overlapping mechanisms that may be common to bothcancer and aging. The loss of the efficient DNA repair capacity is amajor factor in both cancer progression and the aging process. One modelfor aging states that it is the result of accumulation of damage in theDNA genome with resulting loss of function of critical genes. It hasbeen proposed that during the aging process, robust DNA repair eventsbecome less active or inefficient resulting in accumulation of damagedDNA, and eventually in death. One unifying concept simply states thatfailure to repair DNA damage in protooncogene or tumor suppressor genescauses loss of growth control and cancer. However if the accumulation ofDNA damage does not involve these growth regulatory genes this simplyleads to cell death or senescence.

Metalloproteinases: Targets for Pharmacological Therapy

[0080] The mechanisms by which numerous chelating agents work onspecific metalloenzymes have been characterized. These mechanismsprovide investigators with several metalloprotein targets. Specificdrugs can be created that will inactivate the target metalloproteins.This patent application describes a group of novel chelating compoundsthat were designed for the specific control and inactivation ofmetalloproteinases.

[0081] Proteinases are enzymes whose function is the cleaving of proteinchains at specific sites. They play a critical role in the physiology ofviruses, prokaryotic and eukaryotic cells. Proteinases are essential forthe processes of growth, would healing, tissue remodeling, immunologicaldefense, digestion, apoptosis, and coagulation. Pathological activationor inactivation of these enzymes leads to numerous disorders thatcontribute to disease initiation and progression. These enzymes aretargets for the development of proteinase inhibitors which result innumerous drugs for the treatment of diseases such as hypertension,coronary artery disease, asthma, inflammation, arthritis, cancer,metastasis, infectious diseases, cardiovascular, respiratory andneurodegenerative disorders.

[0082] Proteolytic enzymes are able to cleave peptide bonds and arecharacterized as hydrolases. Proteinases are further divided into fourclasses: serine proteases (I), cysteine proteases (II), asparticproteases (III), and metalloproteinases (IV). The compounds of thisinvention refer exclusively to metalloproteinases and are proteinaseinhibitors pharmacologically active at the metal ion containing activeenzymatic site.

[0083] Modification of abnormal metalloproteinase activity represents anopportunity for controlling the initiation and progression of manydiseases. The inventors and others have recognized that effectiveinhibitors of zinc containing metalloproteinases must have at least: 1)one functional group capable of binding to the catalytic zinc such ascarboxylic acid, thiol, or hydroxamic acid; 2) have at least onefunctional group which can H-bond with the enzyme backbone; and 3) haveone or more side chains capable of favorable London interactions withthe enzyme active site. There are numerous examples of specific drugsthat can be used to inhibit zinc metalloenzyme in accordance with theprinciples delineated above, for the purpose of controlling theinitiation and progression of specific diseases. For example, a primeenzyme target in ocular hypertension is carbonic anhydrase (CA). CAs aremetalloenzymes and many inhibitors of these enzymes are metal complexinganions that coordinate directly to zinc in the enzyme active site.Carbonic anhydrases are inhibited by sulfonamides that bind zinc and inthe process substitute a catalytically important water molecule. Anotherexample is the angiotensin-converting enzyme (ACE), a component of therenin-angiotensin system. ACE is a zinc metalloenzyme that is inhibitedby zinc chelators. For example, Captopril was designed to compete withangiotensin for the zinc ion in the enzyme and binds to zinc through athiol group. Lisinopril, another ACE inhibitor binds to zinc through anamino carboxylate moiety. Based on these pharmacological concepts, theinventors present in this application examples of the successful orpromising pharmaceutical applications of a novel type ofmetalloproteinase inhibitors.

Metal Complexes of Chelating Agents as Enzyme Inhibitors

[0084] In recent years major advances in elucidating the interactionbetween metalloproteins and therapeutic agents have allowed exactpredictions for the drug binding sites. The exact nature of thisinteraction is critical to control drug specificity which in turn leadsto the reduction of unwanted side effects. Structural information can beused to design molecules that bind to specific targets. The substratespecificity of enzymes allows the design of drugs with a well-definedspecificity. Metalloenzymes are targets for inorganic drugs since metalsplay a key structural and catalytic role for numerous enzymes such asthe zinc metalloenzymes that are one of the topics of this patentapplication. Neutralizing a metal that is essential to enzymatic actionby another catytically incompetent metal can result in an inactiveenzyme. This pharmacological action can be obtained by the coordinationof an exogenous toxic metal to a specific chelating agent which willresult in the substitution of the metal, or removal of the metal fromthe enzyme active site. The platinum drugs are one example in which thetherapeutic effect of the drug is due to platinum while the chelatingagent ligand is merely a carrier. Metal complexes of carbothioamides andthiosemicarbazones have also been found to inhibit ribonucleotidereductase and possess anticancer activity. Copper complexes and coppercomplexes of anti-inflammatory drugs have been shown to be effective asantiiflammatory agents. Furthermore, the matrix metalloproteinasematrilysin, a zinc enzyme, has been shown to be inactivated by cadmium,which forms an inactive Cd/Zn hybrid. It is evident from these worksthat complexes of metal ions can be very useful in many differentdisease conditions.

[0085] The coordination ability of metals allows the formation of strongattachments through covalent and ionic bonds. The term “metal complexes”is interpreted to include complexes, compounds or ions. This compoundsexert their therapeutic effect by binding to a metal site in an enzymeactive site. The formation of the metal complex is central to theirinhibitory actions.

[0086] Viruses utilize a discrete set of unique enzymes to perform theirlife cycles. Thus, these enzymes provide targets for antiviral drugs.Thiosemicarbazones have been used as antiviral agents.Thiosemicarbazones are also known to inhibit ribonucleotide reductase,RNA-dependent DNA polymerase, and dihydrofolate reductase. HIV-1protease is an aspartyl protease that generates mature proteins from theproducts of the gag and pol genes. Many metal ions have been found to beinhibitors of HIV-1 protease. These observations together with the novelcompounds of this invention may lead to the design of potent inhibitorsof HIV-1 proteases which contain metals that can bind to the enzymethrough ionic or covalent interactions and thus can be strongerinhibitors than the currently available.

Conclusions and Future Prospects From the Evidenced Reviewed in theBackground of the Invention

[0087] Form the foregoing it appears that it would be beneficial to havea product that can interfere with the structure or action of certainzinc finger proteins or zinc metalloproteinases to stop the progressionof certain infectious diseases, proliferative diseases,neurodegenerative diseases, and other diseases that depend upon zincfinger proteins and/or zinc metalloproteinases for the generation of thedisease state. Furthermore, it would be beneficial to provide a productthat can control these diseases by chelating metal ions fromzinc-dependent, iron-dependent or copper-dependent proteins, enzymes,and/or hormone receptors necessary for the initiation, progression andmaintenance of replication of cancer cells and other proliferativedisease conditions. Likewise, it would be beneficial to inhibitangiogenesis in certain disease states, such as cancers, allograftrejection, retinopathies, and post-ophthalmic surgery.

[0088] It is evident that enzymes are natural targets for inorganicdrugs since metals play a key enzymatic role for many enzymes, such asthe zinc metalloproteinases. The coordinating ability of metals holdsthe attractive promise of forming stronger attachments. Thus, it wouldbe beneficial to provide metal complexes that show effectiveness byproviding inhibitory metal ions with the chelating agent merely as acarrier. Furthermore, since the products of this invention are chelatingagents, they may be used to treat metal toxicity conditions such asiron, mercury or lead toxicity.

[0089] With the advent of detailed data banks and highly sophisticatedmolecular modeling, the design of organic-chelate-metal inhibitors hasachieved a great level of precision in targeting viral enzymes. Inaddition, the advent of more detailed modeling techniques about enzymestructures has allowed rigorous characterization of drug-enzymeinteractions. In this invention, we have sought specific targets whichplay key roles in cancer cell metabolism, which are unique to cancercells or are differentially expressed in cancer cells. Likewise, weselected other metalloprotein targets which would be beneficial to stopthe progression of other diseases such as inflammatory diseases.

[0090] It is evident from the published works that the role of metalions in neurodegenerative diseases is complex. The novel agents of thisinvention can provide the penetrability in the CNS, the appropriateconcentration range, and the right pharmacokinetic characteristics forprolong treatment which is required for these diseases. Treatment forthese disorders tend to be long term rather than single dose, and thenovel compounds of this invention can afford specific interactions withcritical enzyme targets and metalloproteins involved in diseaseprocesses. This would separate regulatory processes form toxic effectsof the pharmaceutical agents.

[0091] It is clear from the diversity of therapeutic applications ofchelating agents described in the background of this invention thatdisease pathology correlates with abnormal metalloprotein and/ormetalloenzyme activity and that both metalloprotein and metalloenzymeinhibition can be a powerful and versatile tool in the treatment ofvarious diseases as it will be demonstrated later for the novel agentsof this invention.

SUMMARY OF THE INVENTION

[0092] Based upon the foregoing, several classes of compounds have nowbeen discovered which can be used to inactivate viruses, pathogenicprokaryotes and pathogenic eukaryotes cells. Moreover, theidentification of selected target proteins in these organisms are alsodescribed. The compounds identified are either lead compounds for thedevelopment of drugs or candidates for antiviral, antifective, and othertherapeutic uses. Not every compound showing reactivity with the targetmetalloprotein will be able to penetrate the virus or cells and attackthe target metalloprotein, in addition some will be toxic. However,identifying specific chemical groups that can react with these proteinsenables rational drug design.

[0093] This invention relates to the prophylactic and therapeutictreatment of a mammal hosting a pathogenic virus, prokaryotic pathogenicorganism, or eukaryotic pathogenic cell by the systemic administrationof compounds having the following formula

[0094] Wherein R1, which can be singly or multiply substituted in anyposition of the thiophene ring not already substituted by R2, isselected from the group consisting of hydrogen, alkyl and substitutedalkyl wherein the alkyl portion is from 1 to 21 carbon atoms, inclusive,and isomeric forms thereof, cycloalkyls and substituted cycloalkyl,substituted oxygen, substituted nitrogen, halogen, phenyl, andsubstituted phenyl, —(CH₂)_(n)—OH, —(CH₂)_(n)—NR3R4, and isomeric formsthereof, wherein n is an integer of from 1 to 21, inclusive, R3 and R4are H or alkyl of from 1 to 21 carbon atoms, inclusive, and isomericforms thereof; wherein R2, which can be singly or multiply substitutedin any position of the thiophene ring not already substituted by R1, is

[0095] and X is the amino function of a compound selected from the groupconsisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof, and to the useof compounds of the formula

[0096] Wherein A, B, and D are selected from the group consisting ofnitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim1, and can be attached to any ring carbon or nitrogen atom, the R1 canbe multiply attached to any ring carbon atom; the R1 can be2-carboxylic, 2-hydrazine, 2-hydrazone, and 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof.

[0097] Compounds of particular importance for the subject of thisinvention are the of the following formula:

[0098] wherein R1 is in the 4-position and is a fatty acid of from 1 to21 carbon atoms, inclusive, and isomeric forms thereof; wherein R2 is inthe 3 or 5 position and is an halogen; and the pharmaceuticallyacceptable acid-addition salts thereof, to a mammal hosting a pathogenicorganism or diseased cells.

[0099] Other compounds of particular importance for the subjectinvention, derived from the formula defined immediately above, have thefollowing formulas:

[0100] 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine,4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone.

[0101] Furthermore, within the scope of the subject invention are theuse of compounds of the formula

[0102] Wherein A, B, and D are selected from the group consisting ofnitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim1, and can be attached to any ring carbon or nitrogen atom, the R1 canbe multiply attached to any ring carbon atom; the R1 can be2-carboxylic, 2-hydrazide, 2-hydrazone, and 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof.

[0103] The chemistry and synthesis of one of the analogues describedabove can be described in exemplary form as shown in FIG. 2. Thisanalogue have been shown to be 7-10 times more active than2-thiophenecarboxylic acid against cancer cells in tissue culture.

[0104] More particularly, in one embodiment of the present invention, amethod is provided for dissociating a zinc ion (or copper, or iron orother transition metal ion [TMI]) from zinc finger proteins ormetalloproteinases, the method comprising contacting the metalloproteinwith a compound selected from the group consisting of:

[0105] 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide,4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone orderivatives thereof.

[0106] A method for inactivating a metalloprotein or metalloenzyme of apathogenic virus, pathogenic prokaryotic organisms or pathogeniceukaryotic cells, wherein the said protein comprises an amino acidsequence structure which chelates a zinc ion or a transition metal ion,said method comprising the step of contacting intravirally orintracellularly the said zinc ion or transition metal ion bound to thechelating protein structure, with a chelating compound which dissociatesthe metal ion protein complex selected from the group consisting of thefollowing compounds:

[0107] Examples of such compounds include, but are not limited to thefollowing:

[0108] 1. Furoic acid having the formula described above;

[0109] 2. 2-Thiophenecaboxylic acid having the formula shown in FIG. 1;

[0110] 3. Halogenated furoic acid as defined in claim 1;

[0111] 4. Halogenated 2-thiophenecarboxylic acid as defined in claim 1;

[0112] 5. Hydrazides of furoic acid or 2-thiophenecarboxylic acid havingthe formula —C—NH—NH—R, where —C is attached to position 2 of thethiophene ring;

[0113] 6. Hydrazones of furoic acid or 2-thiophenecarboxylic acid havingthe formula —CH═N—NH—C=0-, where —C is attached to position 2 of thethiophene ring;

[0114] 7. Thiosemicarbazones having the formula —CH══N—NH—C═S—, where Cis in position 2 of the thiophene ring;

[0115] 8. Halogenated furoic acid, halogenated 2-thiophenecarboxylicacid, and derivatives thereof where the halogen is selected from thegroup consisting of F,I,Br, and Cl;

[0116] The compounds of 1 to 8 in which one or more of the ring residuesin positions 3 or 4 have been replaced by a fatty acid side chain of 2to 21 carbons.

[0117] The compounds of 1 to 8 coordinately complexed to cupric ions, orto ferric ions, or to a toxic metal ion such as platinum.

[0118] A method for inactivating a metalloprotein or metalloenzyme of apathogenic virus, pathogenic prokaryotic organisms or pathogeniceukaryotic cells, wherein the said protein comprises an amino acidsequence structure which chelates a zinc ion or a transition metal ion(TMI), said method comprising the step of contacting intravirally orintracellularly the said zinc ion or TMI bound to the chelating proteinstructure with a chelating compound which dissociates the metal ion fromthe protein complex, said chelating compound selected from the groupconsisting of the following compounds:

[0119] 2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid;3,5-dibromo-2-furoic acid;

[0120] 3,4,5-tribromo-2-furoic acid;2,5-dimethoxytetrahydro-2-furoicacid hydrazine;

[0121] 5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;

[0122] 5-[3-(trifluoromethyl)phenyl]-2-furoic acid;

[0123] 5-(2-Nitrophenyl)-2-furoic acid;

[0124] 5-(3-Nitrophenyl)-2-furoic acid;

[0125] 5-(4-Chloro-2-nitrophenyl)-2-furoic acid;

[0126] 5-(4-Chlorophenyl)-2-furoic acid;

[0127] 5-(4-Methyl-2-nitrophenyl)-2-furoic acid;

[0128] 5-(4-Nitrophenyl)-2-furoic acid;

[0129] 5-Bromo-2-furoic acid;

[0130] 5-Chloro-2-furoic acid;

[0131] 5-Nitro-2-furoic acid

[0132] 5-Nitrofuran-2-carboxylic acid.

[0133] 2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylicacid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acidhydrazone;

[0134] 3-methyl-2-thiophenecarboxylic acid;

[0135] 5-bromo-2-thiophenecarboxylic acid hydrazone;

[0136] 5-methyl-2-thiophenecarboxylic acid;

[0137] 5-chloro-2-thiophenecarboxylic acid;

[0138] and 5-chloro-2-thiophenecarboxylic acid hydrazone.

[0139] In yet another embodiment of the present invention a method forselecting a compound capable of dissociating a zinc chelated with a CCCC[or a CHCH, or other permutations of C and H] zinc finger of a viralprotein is as follows: a) contacting the CCCC zinc finger of the CCCCprotein with a chelating agent; and b) detecting the dissociation of thezinc atom from the protein. Such chelating agents include:

[0140] 2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid;3,5-dibromo-2-furoic acid;

[0141] 3,4,5-tribromo-2-furoic acid;2,5-dimethoxytetrahydro-2-furoicacid hydrazine;

[0142] 5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;

[0143] 5-[3-(trifluoromethyl)phenyl]-2-furoic acid;

[0144] 5-(2-Nitrophenyl)-2-furoic acid;

[0145] 5-(3-Nitrophenyl)-2-furoic acid;

[0146] 5-(4-Chloro-2-nitrophenyl)-2-furoic acid;

[0147] 5-(4-Chlorophenyl)-2-furoic acid;

[0148] 5-(4-Methyl-2-nitrophenyl)-2-furoic acid;

[0149] 5-(4-Nitrophenyl)-2-furoic acid;

[0150] 5-Bromo-2-furoic acid;

[0151] 5-Chloro-2-furoic acid;

[0152] 5-Nitro-2-furoic acid

[0153] 5-Nitrofuran-2-carboxylic acid.

[0154] 2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylicacid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acidhydrazone;

[0155] 3-methyl-2-thiophenecarboxylic acid;

[0156] 5-bromo-2-thiophenecarboxylic acid hydrazone;

[0157] 5-methyl-2-thiophenecarboxylic acid;

[0158] 5-chloro-2-thiophenecarboxylic acid;

[0159] The target proteins include but are not limited to: 1) MPS-1/S27ribosomal protein; 2) the viral proteins described in Table 2; 3) LFprotein of the Anthrax bacteria; 4) amyloid beta monomeric precursors ofAlzheimer's disease; and 5) matrix metalloproteinases. The selectionmethod utilizes detection techniques such as: Nuclear Magnetic Resonance(NMR), high pressure liquid chromatography (HPLC), capillaryelectrophoresis, immunoblotting, release of radioactive isotope of metalion, detecting fluorescence, and detecting mobility changes in gel shiftassays.

[0160] It is among the objects of the present invention to providespecifics compounds which can inhibit the growth and proliferation ofcells by blocking the activity of metal ion-containing proteins andmetalloproteases.

[0161] Another object of the present invention is to provide a compoundwhich can retard the growth and proliferation of target infectiveorganisms including viruses, bacteria, fungi, parasites or otherinfective agents by blocking the activity of metal ion-containingproteins.

[0162] Another object of the present invention is to provide a compoundwhich can retard the growth and proliferation of target viruses or virusinfected cells by blocking the activity of transition metalion-containing protein structures such as zinc finger proteins ormetalloenzymes associated with viral replication.

[0163] Another object of the invention is to provide such a compound toretard angiogenesis in malignant tumors by inhibiting metalloenzymesassociated with neovascularization.

[0164] It is still another object of the present invention to provide acompound that can retard the growth of premalignant and malignant cellssuch as virally, chemically and spontaneously transformed cells.

[0165] Another objects of the present invention is to provide a compoundthat can retard the growth of premalignant and malignant cells such asvirally, chemically and spontaneously transformed cells and beadministered by any acceptable route, including orally, with substantialeffectiveness and minimal side effects.

[0166] It is also among the objects of the present invention to providea new treatment for patients suffering from various forms of viralinfections by utilizing the novel properties of metal chelating agentsas a chemotherapeutic anti-viral agent.

[0167] It is another object of the present invention to provide an agentthat can halt the proliferation and transmission of viruses containingzinc finger proteins or metalloproteinases as essential viral proteins.

[0168] Another object of the invention is to provide a method ofdisrupting the function of metal containing protein structurescontaining metals other than zinc, such as iron-finger and otherproteins with metal binding motifs heretofore unidentified by theadministration of a metal chelating agent, both topically andsystemically

[0169] Another object of the invention is to provide a product which canbe spray in the nostrils or inhaled to prevent or control upperrespiratory diseases such as influenza, rhinoviruses or pulmonarycancer.

[0170] Another object of the invention is to provide ananti-inflammatory compound that is effective in a broad range ofinflammatory disorders including inflammatory response to infections andto chemical damage or radiation including, but not limited to,ultraviolet, atomic or medical radiation.

[0171] Yet another object of the invention is to provide such chelatingagents in a relatively safe and nontoxic form such as2-thiophenecarboxylic acid, its derivatives or related or similarcompounds for both topical and systemic use.

[0172] Another object of the invention is to provide a topicalpreparation of metal chelating agents such as furoic acid,2-thiophenecarboxylic acid (2-TH) or its derivatives to treat virallyinduced or spontaneous proliferative diseases of the skin or mucousmembranes in human and animal subjects.

[0173] It is still another object of the present invention to provide anintravaginal preparation containing metal chelating agents such asfuroic acid, 2-TH acid, or derivatives thereof that can prevent orretard sexually transmitted diseases caused by viruses or othercausative agents containing zinc finger proteins or other zinc bindingmotif in their structure.

[0174] Still another object of the present invention is to provide apreparation containing chelating agents such as furoic acid, 2-TH acidor derivatives thereof that halts the progression of viral infections orproliferative diseases that is non-toxic to normal cells, relativelyinexpensive and well suited for its intended purposes.

[0175] According to the invention, the main object is to provide amethod of treatment and compound used in the method, for example, metalchelating compounds, such as 2-thiophenecarboxylic acid or derivativesthereof, for the treatment of infective or proliferative diseases,inflammatory responses, and cancers in human and animal subjects. Theinvention can be used orally or topically to treat or control a widespectrum of proliferative diseases or conditions, both spontaneous orinduced by viruses, bacteria, fungi parasites, chemicals, or radiation.The metal chelating compounds bind metal, for example iron or transitionmetal ions such as zinc, required by enzymes, or by transcriptionproteins found in viruses or malignant cells. By way of further example,the metal chelating compound, for example 2-thiophenecarboxylic acid orits derivatives, is used to bind the zinc contained in the zinc fingerprotein M1 common to the influenza viruses strains, thereby inactivatingthe virus and preventing the exit of RNA containing viruses or particlesfrom the cells.

[0176] In one embodiment of the invention 2-TH acid in 500 mg capsulesgiven in dosages ranging from 500 mg per day to 2000 mg per day, or morehas been demonstrated by mathematical modeling to be effective inreducing the size of tumors, such as cancerous lymph nodes and inducingapoptosis in the cancerous tumor cells.

[0177] One embodiment of a topical preparation consists of a solution ofthe chelator, for example, 0.01% to 99%, preferably 5% to 25%, furoicacid or 2-TH acid in an appropriate vehicle, such as deionized water,buffer or other solvent, and is applied to the lesion three times a day.The preparation can be applied to skin to control acne, warts, herpesinfections and to toe nails, to treat fungal infections. In anotherembodiment, the topical preparation consists of an ointment or creamcontaining approximately 0.5% to 99%, preferably 5% to 10% of furoicacid or 2-TH acid which is applied once or twice daily to the lesion.The ointment or cream can be instilled intravaginally to retard sexuallytransmitted viral diseases.

[0178] The various embodiments of the topical preparations can be usedto treat papilloma and herpes viral diseases and to retard thepapilloma, herpes and HIV viruses as well as proliferative diseases suchas psoriasis and skin cancer.

[0179] Various derivatives that maintain their activity and stabilityafter systemic administration are provided. Slow release oralformulations can be used to treat diseases for the digestive tract. Theactive derivatives can be administered orally, parenterally, byinhalation, transdermally or by any other appropriate method to controlproliferative diseases, cancers, viral infections, HIV, and pulmonaryAnthrax or any other condition wherein the causative agent includes azinc-containing protein, whether the zinc-containing protein is a zincfinger protein, a zinc ring protein, or other type of zinc or metalcontaining structure heretofore unidentified or undetected, wherein themetal containing segment is required for protein stability andconfiguration and/or enzymatic activity.

[0180] It will be appreciated that other appropriate chelating materialssuch as the derivative of 2-TH acid may be used. It also will beappreciated that, although 1% to 5% topical preparations of furoic acidand 2-TH acid are described, a broader range of concentrations may beused. Further, the systemic doses may be altered or adjusted to rangesgreater or lesser than those described, depending on toxicity andpatient response, without departing from the scope of the appendedclaims.

[0181] It will be appreciated that some of the hydrophobic compounds ofthis invention can act intracellularly at low concentrations (pM to uM).Thus, the agents of this invention can penetrate cells, reach the targetmetalloprotein which is present at low intracellular concentrations (uM)and inactivate it.

[0182] It will be also appreciated that while the dose-responserelationships of the chelating drugs of the prior art in general have asharp square wave for the dose-response relationship and arenon-specific, the compounds of the instant invention are target specificand have dose-response relationships for systemic use that are sigmoidaland thus have a wide range of therapeutic concentrations. Therefore, theagents of the instant invention are highly compatible with acceptabletarget to background therapeutic/toxic ratios.

[0183] Other features, objectives and advantages of the invention andits preferred embodiments will become apparent from the detaildescription which follows. Table 1 summarizes some of the medicalapplications of the agents of this invention. TABLE 1 The agents of thisinvention: Specific Chelating Agents and Metal Complexes of these Agentsas Therapeutic Agents to Disrupt Target Zinc Finger Protein and toInhibit Target Metalloenzymes I. Applications to Normal and AbnormalPhysiology Cancer Angiogenesis Proliferative diseases (e.g. psoriasis)Hypertension Matrix Metalloproteinases Neurodegenerative diseases (e.g.Alzheimer's; Parkinson's disease) Inflammation (e.g. Arthritis) Organtransplant II. Applications to Infections A. Viral (Table 2) B.Nonviral: Bacteria, Fungi, and parasites III. Toxicity ApplicationsHeavy Metals (e.g., depleted uranium, lead nickel, tungsten) Poison Ivydermatitis IV. Miscellaneous Enzymes Leukotriene A₄ hydrolase V.Metal-facilitated Inhibition Copper (II) chelates as inhibitors of HIVproteases

BRIEF DESCRIPTION OF THE DRAWINGS

[0184] In the drawings,

[0185]FIG. 1 shows the chemical structures of 2-thiophenecarboxylicacid, 2-thiophenecarboxylic acid hydrazide;3,4,5-thrichloro-2-thiophenecarboxylic acid hydrazone; and4-butyl-3-chloro-2-thiophenecarboxylic acid thiosemicarbazone.

[0186]FIG. 2 illustrates the chemistry and synthesis of4-butyl-3-chloro-2-thiophenecarboxylic acid hydrazide;

[0187]FIG. 3 illustrates the effects of different concentrations of2-thiophenecarboxylic acid on the growth CHO cells;

[0188]FIG. 4 illustrates the effects of different concentrations of2-thiophenecarboxylic acid hydrazide (HY) on the growth of CHO cells;other compounds tested were: Furan (RAN); thiophene (TF), and furoicacid (FA);

[0189]FIG. 5 illustrates the effects of different concentrations of3,4,5-trichloro-2-thiophenecarboxylic acid on the growth of CHO cells;

[0190]FIG. 6A illustrates the effects 2-thiophenecarboxylic acid onmorphology of CHO cells;

[0191]FIG. 6B illustrates the effects of 2-thiophenecarboxylic acidhydrazide on morphology of CHO cells;

[0192]FIG. 6C illustrates the effects of3,4,5-trichloro-2-thiophenecarboxylic acid acid on morphology of CHOcells;

[0193]FIG. 6D illustrates the morphology of CHO cells growing in acontrol culture without drug additions;

[0194]FIG. 7 illustrates the calculated relationship between watersolubility and cytotoxicity of the novel agents. The higher the watersolubility, the higher is the number of surviving cells. The chemicalname of the agents is: (1) 2-thiophenecarboxylic acid; (2)2-thiophenecarboxylic acid hydrazide; (3) 4-butyl-2-thiophenecarboxylicacid hydrazide; (4) 4-pentyl-2-thiophenecarboxylic acid hydrazide; (5)4-hexyl-2-thiophenecarboxylic acid hydrazide; (6)2-chloro-4-(trifluoromethyl)phenyl-2-furoic acid; (7)4-nitro-2-thiophenecarboxylic acid hydrazide; (8)4,5-diphenyl-2-thiophenecarboxylic acid hydrazide;

[0195]FIG. 8 illustrates a molecular model of a zinc finger proteindenoted metallopanstimulin/S27 ribosomal protein with the zinccoordinately bound to four cysteine residues;

[0196]FIG. 9 is a conceptual folding scheme for a metal-binding peptidedomain of a retroviral zinc finger protein and its disruption by theagents of this invention;

[0197]FIG. 10 is a hypothetical scheme of inhibition of angiogenesis bythe agents of this invention which can inhibit multiple targets such aszinc-, copper-, and iron-dependent metalloproteins and metalloproteaseswhich are involved in proteolysis of the extracellular matrix (1), cellmigration and chemotaxis (2), and cell proliferation (3).

[0198]FIG. 11 is a schematic block diagram of the Lethal Factorpolypeptide component of the anthrax toxin. Lethal Factor (LF) is an 87kDa polypeptide and is the catalytic component of the anthrax toxin.Amino acids 401-776 comprise the catalytic domain. This region containsat least one zinc-binding motif which is involved in the LF proteolyticactivity in macrophages. The definitions of domains is based on3D-structures and sequence alignments using standard algorithms;

[0199]FIG. 12 is a schematic block diagram of the domain organization ofDnaJ heat shock protein. DnaJ is a metalloprotein of 87 kDa which isessential for stimulation of the Hsp70 ATPase activity. Amino acids143-200 region contains one zinc-binding motif which is involved insubstrate binding. The definitions of domains is based on 3D-structuresand sequence alignments using standard algorithms.

[0200]FIG. 13 is a hypothetical scheme of inhibition of chronicallograft rejection by the agents of this invention. The smooth musclecell migration, growth, and the persistent perivascular inflammation canbe prevented by the inhibitory effects of this agents on several targetssuch as matrix metalloproteinases, inhibition of smooth muscle cellmigration, cell division, neointimal formation, inflammation andangiogenesis;

[0201]FIG. 14 illustrates the binding of 4-pentyl-2-thiophenecarboxylicacid thiosemicarbazone (4-P-2-TA-TSC) to the active site structure ofhuman LTA₄ hydrolase. The zinc-binding ligands are His-295, His-299, andGlu-318. The shaded area indicates the L-shape hydrophobic cavity towhich the 4-pentyl residue of 4-P-2-TA-TSC binds. The binding of thethiol group of 4-P-2-TA-TSC to the zinc atom at the active site of LTA₄hydrolase is also shown. 4-P-2-TA-TSC competes with the substrate forthe active site of LTA₄ hydrolase.

DEFINITIONS

[0202] “Penetrating” refers to the act of bringing the drug into anintracellular or viral compartment which will place the active moiety ofthe drug into adequate proximity to the target protein such as thereaction will occur and the target protein will be inactivated.

[0203] “Zinc finger protein disrupting agent” refers to the action of achelating agent that specifically ejects the zinc from the protein,resulting in a conformational change of the protein that renders theprotein inactive.

[0204] “Chelating agent catalytic inhibitor of metalloenzymes” refers tothe inhibitory action of a specific chelating agent that binds to thezinc present in the catalytically active site of the enzyme and rendersthe site catalytically inactive.

[0205] “Organometallic compound” refers to a specific chelating agenttightly bound to a toxic metal that inhibits the active site of ametalloenzyme by replacing the naturally occurring metal ion in theenzyme.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0206] From the foregoing it appears that it would be beneficial to havea product that can interfere with the formation or action of certainzinc finger proteins or metalloproteinases to inhibit the progress ofpathogenic viruses, bacteria, fungus, malignancies, neurodegenerativediseases, etc., dependent upon zinc finger or metalloproteinases fortheir pathogenicity. Furthermore, it would be beneficial to provide aproduct that can halt the growth of proliferative cells, such asmalignant cells by chelating metal ions from zinc-dependent ortransition metal ion (e.g. copper, iron, etc.)-dependent proteins andenzymes necessary for the replication of the malignant cells.Furthermore, since the products can chelate metals, such as iron, theyhave a role in metal toxicity states, such as iron or lead toxicity.

[0207] In view of the foregoing, there exists a need in the art for apathogenic target protein, mechanism and methods which can be used topredict compounds that can effectively disrupt specific zinc-fingers orother zinc-binding motif and, in turn, inactivate the virus, prokaryoticor eukariotic cell of choice. Similarly, there exists a need forcompounds that can be used effectively to inactivate catalytic zincbound to the active site of metalloproteinases and, subsequently,inactivate the virus, prokaryotic or eukaryotic cell of choice.

[0208] The present invention provides a descriptive mechanism for thedisruption of zinc fingers and other conserved structures that containmetal ions essential for the function of metalloproteins. The disruptionis accomplished by several classes of chemical compounds which areidentified by their capacity to react with and disrupt zinc finger andother metalloproteins essential for cellular and viral functions.

[0209] The pharmacological and therapeutic characteristics of the agentsof the instant invention will be described in detail in the followingsections. Data generated from results of in vitro assays, animal models,and computer modeling show the utility of this invention in the medicalfield.

[0210] Although the utility of this invention is multiple, we present afew examples that provide proof of the utility of this invention innumerous medical fields such as cancer, infectious diseases, andneurodegenerative diseases.

[0211] The novelty and non-obviousness of the agents of the instantinvention is demonstrated by the fact that the majority of the chelatingagents utilized prior to the instant invention are too harsh to be usedsystemically in animals and humans even for short time periods. One ofthe main problems with the chelating agents currently available forsystemic treatments is that they lack specificity and have dose-responserelationships that are too sharp to be useful in systemic clinicalapplications. In fact, with prolong use, most of these agents cause abody depletion of transition metal ions essential for cellularrespiration with the consequent development of patient anergia.

[0212] While the dose response relationships of the drugs of the priorart in general have a sharp square wave for the dose-responserelationship and are non-specific, the compounds of the instantinvention are target specific and have dose-response relationships forsystemic use that are sigmoidal and thus they have a wide range oftherapeutic concentrations. Thus the agents of the instant invention arehighly compatible with acceptable target to background therapeutic/toxicratios.

[0213] The present invention provides a descriptive mechanism for thedestruction of virally infected cells and proliferating cancer cellsinduced by the chemotherapeutic agents of this invention. Theexperimental evidence supports the conclusion that zinc deficiencyresulting from exposure of culture cells to membrane-permeableZn²⁺-chelators, can induced apoptosis in virally transformed cells whilenormal cells remain unaffected. Furthermore, iron deficiency resultingfrom exposure of cultured cells to membrane-permeable Fe²⁺-chelators,can induced apoptosis in virally transformed cells while normal cellsremain unaffected. An intracellular pool of chelatable Zn²⁺ plays acritical role in apoptosis, most likely by modulating the activity ofzinc finger proteins and inhibiting the enzymatic activity of zinccontaining metalloenzymes, which are essential for maintenance ofcellular and viral structure and cancer cell proliferation. Theintracellular pool of chelatable Fe²⁺ also plays a critical role inapoptosis, most likely by modulating the activity of Fe^(2+/3+)containing proteins such as ribonucleotide reductase, which areessential for maintenance of viral structure and function and cancercell proliferation.

[0214] The present invention contemplates the use of metal chelatingagents such as furoic acid, 2-thiophenecarboxylic acid (2-TH) and theirderivatives, analogs and related chemical as defined above, in theprevention and treatment of diseases and toxicities caused by heavymetals such as iron, copper, nickel, lead, uranium, and tungsten. Thechelating agent can be administered to the patient in oral form or ininjectable form. Other forms of administration include transdermal,rectal suppositories, intranasally, inhalation, or any otherpharmacologically acceptable form. Chelation therapy with theheavy-metal antagonist of this invention results in the binding of thetoxic metal to the chelating agent. Chelation therapy will promote theexcretion of the inorganic heavy metal. The inactive chelate-heavy metalis then eliminated from the body. Chelation therapy is indicated insymptomatic patients of patients with toxic levels of a given toxicmetal. The agents of this invention can be combined with other chelatorssuch as EDTA, dimercaprol, D-penicillamine and succimer. Whenenvironmental exposure to heavy metals is a concern, the heavy-metalantagonists of this invention can be used to prevent intoxication.

[0215] In this patent application we also present novel organometallicagents that are derivatives of the chelating agents of this invention.It is pertinent to mention here that organometallic compounds have beenused in medicine as antiseptics and antimicrobial for several centuries.These organometallic agents are known to exert their effects byinhibiting the active sites of metalloenzymes. The organometalliccompounds of this invention can carry the metal ion as a toxic agent forthe specific virus, microbe or cancer cell. For example, the inhibitionof zinc metalloproteases in bacteria and fungi is within the scope ofthis invention. Screening a large number of toxic metal ions against themetalloproteases of bacteria and fungi, in the presence and absence ofthe chelating agents of this invention can show an increase in theactivity of the compound tested in the presence of the toxic metal. Thisinhibition is competitive, indicating that the organometallic inhibitorbinds to the active site of the metalloenzyme. For example, trivalentarsenical drugs of this invention may be used in the treatment oftrypanosome-mediated illnesses such as African sleeping disease andChaga's disease. Trivalent arsenical of this invention bind arsenic byinteraction with the compound thiol groups. The mechanism for theiractivity is due to an interaction of arsenic with the parasite targetprotein thiol groups of the active site of the metalloenzyme. Complexesof metal ions can show effectiveness as antifungal agents such as thepathogenic yeast Candida albicans. Another example is Mycloplasm whichlacks a rigid cell wall. The agents of the instant invention can act onmetalloenzymes of Mycoplasm. Furthermore, copper is known to be toxic tomycoplasm. The agents of this invention can be use as copper carriers tocontrol Mycoplasm infection. Understanding better the effectiveness ofthe agents of this invention against infection will also enhance thedevelopment of anticancer and antiviral agents.

[0216] All the applications of the therapeutic drugs of this inventionare designed to combat abnormal microorganisms and abnormal cells whileleaving the surrounding normal tissues intact. In this context, theagents of this invention operate as specific and selective toxins.

[0217] 1. General Methods for the Identification of the Agents of thisInvention

[0218] The present invention provides several classes of compounds whichcan be used to inactivate viruses, prokaryotic and eukaryotic cells. Themethod consists in attacking zinc finger proteins at the zinc fingermotif structural site with the chelating agents of this invention whichwill result in the ejection of zinc, with the consequent disruption ofthe zinc finger protein structure and function. The compounds of thepresent invention can also be used to inhibit catalytically activecenters of metalloenzymes by forming a complex consisting of the activecompound bound to the metal ion present at the catalytic active site ofthe enzyme. This complex formed by the active compound and thecatalytically active metal ion coordinately bound to the amino acids ofthe enzyme active site renders the metalloenzyme inactive.

[0219] In particular, the present invention also describes a set ofspecific tests and reagents that can be used to screen and identifycompounds based on their ability to react with and disruptmetalloproteins. It will be apparent to those skilled in the art thatafter inactivation, the viral protein, or prokaryotic, or eukaryoticprotein so treated, can be used, for example as vaccines or as acomponents in assays for the diagnosis of infections.

[0220] There are several general methods that can be used to detect theactivity of the compounds of the present invention which are brieflydescribed for illustrative purposes only. An example is the release ofradioactive zinc-65-, copper-64-, or iron-59 from labeled protein. Thelabeled protein and precipitating reagents can be used to detect theability of the compounds to remove the radioactive metal from theprotein by determining the bound and free radioactive metal. CapillaryZone Electrophoresis (CZE) can be use to easily detect the activity ofthe compounds interacting with specific metalloproteins. The compoundsthat react with the metalloprotein and remove the transition metalproduce a change in the configuration and charge of the protein whichalters the electrophoretic mobility of the protein with respect to theuntreated metalloprotein. Thus, the electrophoretic mobility of thetreated protein will be different than the control, untreated protein.In addition, the following assays can be used. Gel Mobility shift assayscan be use to detect the activity of the compounds interacting withspecific metalloproteins by detecting changes in protein mobility withand without the agent. Fluorescent zinc chelators can be used to monitorthe release of zinc from the zinc finger or metalloprotein under study.Nuclear Magnetic Resonance (NMR) can also be used to monitor the loss ofzinc or other transition metal from the metalloprotein in the presenceand absence of the compound. By using computer modeling, compounds withspecific properties can also be found by creating three-dimensionalmolecular structures and inspecting the interactions with the activemetal containing sites of the target protein. The drug can also betested for activity by examining its effects on virus infected cells,cultured bacteria and cells. Finally, the drugs can be tested inadequate animal model systems to determine their therapeutic effects andtoxicities.

[0221] There are several general enzymatic methods that can be used todetect the activity of the compounds of the present invention which arebriefly described for illustrative purposes only. For example, thecompetitive inactivation of the zinc metalloenzyme, Leukotriene A₄hydrolase (LTA₄H) by the agents of the present invention can be studiedas follows. The enzyme is competitively inactivated by a structuralisomer of 4-pentyl-thiophenecarboxylic acid hydrazide (P-TAH) thatinteracts with the catalytically active zinc and with the hydrophobicresidues surrounding the catalytically active zinc site. Kineticanalysis shows that the inactivation is directly coupled to catalysisand proportional to product formation. Electrospray mass spectrometrywill show a shift in molecular weight of inactivated enzyme compatiblewith the coupling of LTA₄H in a 1:1 stoichiometry between the drug(P-TAH) and the protein (LTA₄H). The competitive inhibitor P-TAHprevents the covalent binding of LTA₄ to the enzyme, indicating that itoccurs at the active site. This shows the high specificity for the agentof this invention, P-TAH, which is demonstrated by the narrow substratespecificity of LTA₄.

[0222] 2. Compound Chemistry

[0223] Detailed studies of the reaction mechanisms of the drugs to bedescribed here, which include interaction with specific metalloproteins,and the effects of the drugs on cultured cancer cells, presented for thefirst time in this application, reveals that certain chelating compoundshaving the functional groups described in this invention have suitablecharacteristics as chemotherapeutic agents, antiviral agents, andbiological response modifiers

[0224] The present invention provides several classes of compounds whichcan be used to inactivate metalloproteins and metalloenzymes involved inpathological conditions. Compounds which interact with metalloproteinsand metalloenzymes include but are not limited to the following:

[0225] Wherein R1, which can be singly or multiply substituted in anyposition of the thiophene ring not already substituted by R2, isselected from the group consisting of hydrogen, alkyl and substitutedalkyl wherein the alkyl portion is from 1 to 21 carbon atoms, inclusive,and isomeric forms thereof, cycloalkyls and substituted cycloalkyl,substituted oxygen, substituted nitrogen, halogen, phenyl, andsubstituted phenyl, —(CH₂)_(n)—OH, —(CH₂)_(n)—NR3R4, and isomeric formsthereof, wherein n is an integer of from 1 to 21, inclusive, R3 and R4are H or alkyl of from 1 to 21 carbon atoms, inclusive, and isomericforms thereof; wherein R2, which can be singly or multiply substitutedin any position of the thiophene ring not already substituted by R1, is

[0226] and X is the amino function of a compound selected from the groupconsisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof, and to the useof compounds of the formula

[0227] Wherein A, B, and D are selected from the group consisting ofnitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim1, and can be attached to any ring carbon or nitrogen atom, the R1 canbe multiply attached to any ring carbon atom; the R1 can be2-carboxylic, 2-hydrazide, 2-hydrazone, and 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof.

[0228] Compounds of particular importance for the subject of thisinvention are the of the following formula:

[0229] wherein R1 is in the 4-position and is a fatty acid of from 1 to21 carbon atoms, inclusive, and isomeric forms thereof; wherein R2 is inthe 3 or 5 position and is an halogen; and the pharmaceuticallyacceptable acid-addition salts thereof, to a mammal hosting a pathogenicorganism or diseased cells.

[0230] Other compounds of particular importance for the subjectinvention, derived from the formula defined immediately above, have thefollowing formulas:

[0231] 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide,4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone.

[0232] Furthermore, within the scope of the subject invention are theuse of compounds of the formula

[0233] Wherein A, B, and D are selected from the group consisting ofnitrogen, oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim1, and can be attached to any ring carbon or nitrogen atom, the R1 canbe multiply attached to any ring carbon atom; the R1 can be2-carboxylic, 2-hydrazide, 2-hydrazone, and 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof.

[0234] The chemistry and synthesis of one of the analogues describedabove can be described in exemplary form as shown in FIG. 2. Thisanalogue have been shown to be 7-10 times more active than2-thiophenecarboxylic acid against cancer cells in tissue culture.

[0235] More particularly, in one embodiment of the present invention, amethod is provided for dissociating a zinc ion (or copper, or iron orother transition metal ion [TMI]) from zinc finger proteins ormetalloproteinases, the method comprising contacting the metalloproteinwith a compound selected from the group consisting of:

[0236] 4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide,4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. orderivatives thereof.

[0237] A method for inactivating a metalloprotein or metalloenzyme of apathogenic virus, pathogenic prokaryotic organisms or pathogeniceukaryotic cells, wherein the said protein comprises an amino acidsequence structure which chelates a zinc ion or a transition metal ion,said method comprising the step of contacting intravirally orintracellularlly the said zinc ion or transition metal ion bound to thechelating protein structure, with a chelating compound which dissociatesthe metal ion protein complex selected from the group consisting of thefollowing compounds:

[0238] Examples of such compounds include, but are not limited to thefollowing:

[0239] 1. Furoic acid having the formula described above;

[0240] 2. 2-Thiophenecaboxylic acid having the formula shown in FIG. 1;

[0241] 3. Halogenated furoic acid as defined in claim 1;

[0242] 4. Halogenated 2-thiophenecarboxylic acid as defined in claim 1;

[0243] 5. Hydrazides of furoic acid or 2-thiophenecarboxylic acid havingthe formula —C—NH—NH—R, where —C is attached to position 2 of thethiophene ring;

[0244] 6. Hydrazones of furoic acid or 2-thiophenecarboxylic acid havingthe formula —CH═N—NH—C=0, where —C is attached to position 2 of thethiophene ring;

[0245] 7. Thiosemicarbazones having the formula —CH═N—NH—C═S—, where Cis in position 2 of the thiophene ring;

[0246] 8. Halogenated furoic acid, halogenated 2-thiophenecarboxylicacid, and derivatives thereof where the halogen is selected from thegroup consisting of F,I,Br, and Cl;

[0247] The compounds of 1 to 8 in which one or more of the ring residuesin positions 3 or 4 have been replaced by a fatty acid side chain of 2to 21 carbons.

[0248] The compounds of 1 to 8 coordinately complexed to cupric ions, orto ferric ions, or to a toxic metal ion such as platinum.

[0249] A method for inactivating a metalloprotein or metalloenzyme of apathogenic virus, pathogenic prokaryotic organisms or pathogeniceukaryotic cells, wherein the said protein comprises an amino acidsequence structure which chelates a zinc ion or a transition metal ion(TMI), said method comprising the step of contacting intravirally orintracellularly the said zinc ion or TMI bound to the chelating proteinstructure with a chelating compound which dissociates the metal ion fromthe protein complex, said chelating compound selected from the groupconsisting of the following compounds:

[0250] 2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid;3,5-dibromo-2-furoic acid;

[0251] 3,4,5-tribromo-2-furoic acid;2,5-dimethoxytetrahydro-2-furoicacid hydrazine;

[0252] 5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;

[0253] 5-[3-(trifluoromethyl)phenyl]-2-furoic acid;

[0254] 5-(2-Nitrophenyl)-2-furoic acid;

[0255] 5-(3-Nitrophenyl)-2-furoic acid;

[0256] 5-(4-Chloro-2-nitrophenyl)-2-furoic acid;

[0257] 5-(4-Chlorophenyl)-2-furoic acid;

[0258] 5-(4-Methyl-2-nitrophenyl)-2-furoic acid;

[0259] 5-(4-Nitrophenyl)-2-furoic acid;

[0260] 5-Bromo-2-furoic acid;

[0261] 5-Chloro-2-furoic acid;

[0262] 5-Nitro-2-furoic acid

[0263] 5-Nitrofuran-2-carboxylic acid.

[0264] 2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylicacid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acidhydrazone;

[0265] 3-methyl-2-thiophenecarboxylic acid;

[0266] 5-bromo-2-thiophenecarboxylic acid hydrazone;

[0267] 5-methyl-2-thiophenecarboxylic acid;

[0268] 5-chloro-2-thiophenecarboxylic acid;

[0269] and 5-chloro-2-thiophenecarboxylic acid hydrazone.

[0270] In yet another embodiment of the present invention a method forselecting a compound capable of dissociating a zinc chelated with a CCCC[or a CHCH, or other permutations of C and H] zinc finger of a viralprotein is as follows: a) contacting the CCCC zinc finger of the CCCCprotein with a chelating agent; and b) detecting the dissociation of thezinc atom from the protein. Such chelating agents include:

[0271] 2-furoic acid; 2-furoic acid hydrazide; Tetrahydro-2-furoic acid;3,5-dibromo-2-furoic acid;

[0272] 3,4,5-tribromo-2-furoic acid;2,5-dimethoxytetrahydro-2-furoicacid hydrazine;

[0273] 5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;

[0274] 5-[3-(trifluoromethyl)phenyl]-2-furoic acid;

[0275] 5-(2-Nitrophenyl)-2-furoic acid;

[0276] 5-(3-Nitrophenyl)-2-furoic acid;

[0277] 5-(4-Chloro-2-nitrophenyl)-2-furoic acid;

[0278] 5-(4-Chlorophenyl)-2-furoic acid;

[0279] 5-(4-Methyl-2-nitrophenyl)-2-furoic acid;

[0280] 5-(4-Nitrophenyl)-2-furoic acid;

[0281] 5-Bromo-2-furoic acid;

[0282] 5-Chloro-2-furoic acid;

[0283] 5-Nitro-2-furoic acid

[0284] 5-Nitrofuran-2-carboxylic acid.

[0285] 2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylicacid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acidhydrazone;

[0286] 3-methyl-2-thiophenecarboxylic acid;

[0287] 5-bromo-2-thiophenecarboxylic acid hydrazone;

[0288] 5-methyl-2-thiophenecarboxylic acid;

[0289] 5-chloro-2-thiophenecarboxylic acid;

[0290] The target proteins include but are not limited to: 1) MPS-1/S27ribosomal protein; 2) the viral proteins described in Table 2; 3) LFprotein of the Anthrax bacteria; 4) amyloid beta monomeric precursors ofAlzheimer's disease; and 5) matrix metalloproteinases. The selectionmethod utilizes detection techniques such as: Nuclear Magnetic Resonance(NMR), high pressure liquid chromatography (HPLC), capillaryelectrophoresis, immunoblotting, release of radioactive isotope of metalion, detecting fluorescence, and detecting mobility changes in gel shiftassays.

[0291] Other features, objectives and advantages of the invention andits preferred embodiements will become apparent from the detaildescription which follows.

[0292] 3. Administration of Compounds In Vivo.

[0293] The compounds of the present invention, identified as those thatinactivate in vitro specific metalloproteins, penetrate viruses,bacteria and cells, and inactivate in vivo the target metafloproteinscan be used to treat metafloprotein mediated diseases such as AIDS,Anthrax, cancer, etc, thereby inactivating the pathogenic life form.

[0294] The compounds used in the present method of treatment areadministered in any suitable manner alone or with suitablepharmaceutical carriers. There are a wide variety of suitableformulations of the pharmaceutical compositions utilizing the agents ofthe present invention.

[0295] For oral administration, the formulations can consist of thefollowing: 1) liquid solutions utilizing diluents such as water, saline,syrups; 2) tablets, capsules, or powders; 3) suspensions and emulsions.The active components can be administered as aerosol formulations to beadministered by inhalation. For rectal administration suitablesuppository bases including hydrocarbons can be utilized. For parenteraladministration such as intravenous, intramuscular, intradermal,subcutaneous, intraperitoneal, and intraarticular delivery, aqueous andnon-aqueous isotonic buffered sterile injections can be used.

[0296] The doses administered to a patient should be sufficient toproduce a therapeutic response with acceptable adverse side effectswhich will result in the shortening or halting of the disease condition.The dose will be determined by the surface area or body weight of thepatient to be treated and the severity of the condition. To determinethe effective amount of the active ingredient in the treatment orprophylaxis of metalloprotein-mediated diseases such as Anthrax, thepractitioner will evaluate the circulating serum levels, toxicity andresponse to the agent.

[0297] The compounds of this invention can be combined with conventionaltherapies, including cytotoxic agents, antibiotics, and biologicalresponse modifiers.

[0298]FIG. 1 summarizes the know chemistry of the 2-thiophenecarboxylicacid (2-TH) family of compounds. 2-TH and furoic acids can chelatedivalent metal ions, including zinc, iron and copper. FIG. 9 shows thata retroviral zinc finger protein can be inactivated by 2-TH acid. Thereagent attacks the zinc bound to the retroviral zinc finger protein(ZFP) and the reaction proceeds until the ZFP is unfolded andsubsequently degraded by Ca2+ dependent intracellular proteases, leadingto apoptosis. The reaction releases zinc from the proteins and the zincbinds to the chelating agent.

[0299] As mentioned above, there are many families of viruses which aredependent upon metalloproteins having a zinc finger domain in theirstructure for replication of the virus. 2-TH acid, furoic acid or othersuitable derivatives or analogs, can be administered orally to patientsexposed to or suffering from viral diseases to bind the metal in theviral metalloprotein and thereby control the disease.

[0300] A pharmaceutically active and acceptable preparation of 2-TH acidor derivative in a concentration of approximately 1% to approximately99%, preferably in a daily range of approximately 500 mg to 6000 mg,preferably approximately 500 mg to approximately 2000 mg of 2-TH acidcan be used for this mode of treatment. It will be appreciated thatdoses approximating the LD-50 of 30 grams/70 Kg may be covered by theinvention in the event continued research shows higher doses areoptimal.

[0301] Novel substituted derivatives of 2-TH acid and related compoundscan be used systemically to treat cancer, viral infections and otherrelated diseases and proliferative disorders. The novel substitutedderivatives of 2-TH acid and related compounds also work by disruptingthe binding of zinc atoms in zinc finger proteins or metalloproteases orother structures heretofore unknown that depend upon the inclusion ofzinc or other transition metal ions, for stability, packaging, orenzymatic activity. Further, the novel substituted derivatives arestable and retain their zinc chelating properties even when introducedsystemically by injection, oral administration, inhalation ortransdermal or other routes of administration

[0302]FIGS. 1 and 2 illustrate novel derivatives of 2-TH acid forsystemic use. Computer modeling indicates that such derivatives caninteract with zinc atoms and disrupt its binding to the zinc fingerprotein or metalloproteases. Substitutions at positions 3, 4, and 5 onthe 2-thiophenecarboxylic acid have the proper configuration to preventinterference with the zinc finger protein backbone. For example R3, R4,or R5 can be a methyl, ethyl, propyl, is isopropyl, butyl, isobutyl,secondary butyl, tertiary butyl, pentyl, isopentyl, neopentyl or similargroup. Further, substitution with halogens such as fluorine, chlorine,bromine and iodine can result in effective, systemically active agents.The systemic compounds can be prepared by methods generally known to theart and include pharmacologically acceptable salts thereof.

[0303]FIG. 8 illustrates the binding of zinc in a zinc finger proteindenoted metallopanstimulin. Further, as shown in FIG. 14, the4-pentyl-2-thiophenecarboxylic acid thiosemicarbazone (4-P-2-TA-TSC) canattach to both the catalytic zinc and the amino acids on a hydrophobicpocket of the enzyme LTA₄ hydrolase forming a ternary complex comprisedof the protein, the zinc, and the 4-P-2-TA-TSC acid derivative whichinactivates the enzyme. Therefore, the above-listed moieties that can besubstituted at various positions in the furan ring of 2-TH acid canresult in a 2-TH acid derivative that not only is more stable forsystemic administration, but also one that has even greater affinity andspecificity for, and binding potential with, various zinc fingerproteins or metalloproteinases.

[0304] It will be appreciated that substitutions at the 3, 4, and 5positions can be made with an amino acid or a peptide of two totwenty-one amino acids or more with either basic or acid amino acidspredominating. The substituted 2-TH acid would have an increasedmolecular weight and a substantially increased half-life in the blood.Further, such compounds would penetrate the cancer cells orvirus-containing cells more effectively due to the amphipathic nature ofthe peptide residues.

[0305] The systemic compounds can be administered to human and animalsubjects by any means that produces contact of the active agent with thetarget protein, such as orally, parenterally, inhalation, transdermally,rectally, on any other method for obtaining a pharmacologicallyacceptable blood level. In general, a pharmacologically effective dailydoes can be from about 0.01 mg/kg to about 25 mg/kg per day or any otherpharmacologically acceptable dosing. A pharmaceutically active andacceptable preparation of 2-TH acid or derivative in a concentration ofapproximately 1% to approximately 99%, preferably in a daily range ofapproximately 250 mg to 6000 mg, preferably approximately 500 mg toapproximately 2000 mg of 2-TH acid can be used for this mode oftreatment. It will be appreciated that doses approximating the LD-50 of30 grams/70 Kg may be covered by the invention in the event continuedresearch shows higher doses are optimal.

[0306] It will be appreciated that in vivo administration of 2-TH acidor its derivatives for the treatment of cancer, for example, hasunexpected results in animals, not predicted by the effect of furoicacid or 2-TH acid on cells in vitro, as described below in the examples.The inventors have determined that in vitro and in vivo, the compoundsenhance the activity of macrophages.

[0307] The products, such as 2-TH acid, 4-butyl-2-TH-acid andderivatives, may be used to removed metals from a subject in diseasestates such lead poisoning or radioactive contaminations. Oral doses orinjectable doses in the broad range of 250 mg to less than 30 grams perday may be used. Optimally, a dosage of 500 mg to 2000 mg per day wouldbe used, with dosages up to 6000 mg or more in resistant cases.

[0308] The claimed invention is intended to include any other chemicalcompounds, either derivatives of 2-TH acid, compounds with structuralrelationships to 2-TH acid that function to chelate, attach to, ormodify metal ions in proteins structures, including, but not limited totransition metal ions found in proteins structures of viruses,proliferative cells (plant or animal) or even as components of fungi andbacteria.

[0309] 4. Therapeutic Applications

[0310] The breath and depth of the applications of the agents of thisinvention as enzyme inhibitors in biomedical applications is extensive.It is evident from the diversity of therapeutic application presented inthis patent application that pathogenesis correlates with abnormalmetalloprotein expression or abnormal metalloenzyme activity. Theinhibition of metalloproteins or metalloenzymes can be a powerful andversatile tool in the treatment of seemingly unrelated diseases. Table 1shows the potential extensive use of the agents of this invention.

[0311] There are a number of points addressed in this invention thatallows the realization of the full potential of this agents as chelatorsor as metal complexes of the chelators. These factors, which are commonto any therapeutic agent include: Specificity, biovailavility,compensatory effects in vivo, and stability. Specificity is a criticalgoal in the design of therapeutic applications of the compounds of thisinvention. Drugs generally exert some type of toxic effect andnonspecific events can lead to side effects. One of the attractions oftargeting drugs to active sites of enzymes for their particularsubstrate is specificity. To determine selectivity tests of the compoundagainst representative members of potential cross-reactive families ofmetalloenzymes should be done. The methods of this invention allow tocreate agents with truly demonstrated specificity and selectivity. Foroptimum effect, an inhibitor must be readily available at the targetsite. The inhibitor should reach the metalloprotein, anywhere in thecell. For example, if the enzyme is cytoplasmic, the inhibitor mustcross the plasma membrane to reach the metalloenzyme. Thecharacteristics of the side chains of the compounds of this inventionallow high cellular penetrability. The same groups that allow cellularpenetrability are the groups that specifically and selectively interactwith the hydrophobic pockets surrounding the catalytically active metalsite. Of course, in certain instances such as bacteria, fungus, andalgae, this may require a delivery vehicle if the agent itself isnon-membrane permeable. Although the agents of this invention arespecific and efficient in inhibiting a single enzyme in vitro, it can bedifficult to predict its ultimate effect in vivo. For example, aspecific inhibition of an enzyme may result in unanticipated changes inenzymes of another pathway. It is unlikely that this will be the case inthe therapeutic applications of our compounds because the proteins to beinhibited are the ones that are pathogenic and have to be antagonized tocreate a healthy state. Finally, the complexes of chelating agent/metalion as a therapeutic combination must be exceptionally stable aschemotherapeutic agents in vivo and the metal should only be released atthe target site.

[0312] Novel substituted derivatives of 2-TF acid and related compoundscan be used systemically to treat cancer, viral infections,proliferative disorders, bacterial infections, parasitic diseases andother diseases that utilize metalloproteins as critical components ofthe pathogenic effect. The novel substituted derivatives of 2-TF acidand related compounds have a similar mechanism of action as the leadcompounds. The mechanisms of enzyme inhibition and proteinneutralization induced by this compounds relates to the high specificityof the agents to disrupt the binding of zinc atoms present in structuralsites of metalloproteins or to inactivate the zinc containing catalyticsite of metalloenzymes. As a consequence of disruption of the zinc ionsor inhibition of its function by the specific chelating agent, thespecific function of the protein is eliminated. For example, zinc ionsrequired for protein stability, packaging of virus RNA, cellularreplication, etc will be render ineffective. Moreover, the novelsubstituted derivatives are stable and thus, able to perform thespecific functions when introduced systemically by injection, oralroute, inhalation, rectally, or transdermally.

[0313] The invention will be described in additional detail bypresenting specific examples. The following examples are described indetail for illustrative purposes only and they are not intended to limitthe invention in any manner.

[0314] Examples of the specific effects of metal chelating agents,including 2-TH acid and substituted 2-TH acid derivatives thereof, aswell as the practical application of those agents will now be described:

Pharmacodynamics Mechanism of Drug Action and the Relationship BetweenDrug Concentration and Effect

[0315] In this section of this application we summarize the biochemicaland physiological effects of the drugs and their mechanism of action.Moreover, the objective of this section is to characterize thepharmacodynamic differences between the chelating agents of thisinvention and other chelating agents previously used. Such analysisprovides the basis for both the rational therapeutic use of the drugs ofthe instant invention and the design of the new and superior drugs basedon the data present in this application.

[0316] The affinity of the drugs for its receptor and its intrinsicactivity are determined by its chemical structure. The term receptor isoperationally used to denote any zinc finger protein (ZFP) ormetalloenzyme target to which the drug binds specifically to initiateits effects.

[0317] As it will be shown later, relatively minor changes in the drugmolecule results in major beneficial changes in pharmacologicalproperties. The drugs develop here have the followingcharacteristics: 1) A more favorable ratio of therapeutic to toxiceffects than other chelating agents; 2) enhanced selectivity amongdifferent cells as exemplify by the differential effects in normalversus cancer cells; 3) more cell penetrability than those of otherchelating agents; and 4) they can be modified in a way in which they canbe made specific and selective for the specific target metalloenzyme ortarget ZFP.

[0318] One of the basic differences between our drugs and other commonlyused chelators is in the dose-response curves, a representation of theobserved effect of a drug as a function of its concentration when itinteracts with its specific receptor. The chemical affinity of our drugsfor its receptor is in the appropriate range to modify the physiology ofthe specific target metalloenzymes or target ZFP receptor proteins.Thus, the drugs of this invention are specific and effective and havespecific receptor-protein targets.

[0319] As shown by many examples in the literature (Fernandez-Pol,2001), the shape of the curve for typical chelating agents such ascarboxylic acids, its derivatives or EGTA, show a sharp dose-responserelationship (ID₅₀ in the mM range) indicating very harsh effects on thecells, compatible with non-specific cytotoxicity to numerousmetalloproteins. In contrast, the sigmoidal shape of the curves for theselected agents of this invention (FIG. 4), show that these agents havea wide concentration range (ID₅₀ in the nM to uM range), compatible withacceptable or no toxicity to normal cells. Thus, the differentefficacies of the cellular stimulus-response for the various types ofchelating agents previously utilized (Fernandez-Pol, 2001) compared withthe novel chelating agents presented here show the superior nature ofthe agents of this invention.

[0320] The inventors have recognized the properties of 2-TH acid andderivatives as antiproliferative agents by in vitro studies with bothnormal and cancer cells. 2-TH acid, is a metal chelating compound thatinhibits the growth of numerous cultured normal and transformed cells.It has been shown that 2-TH acid can arrest prokaryote and eukaryotecell growth by inhibition of zinc and iron requiring enzymes. Incontrast to its inhibitory activity in proliferating cells, 2-TH acidhas a number of biological properties such as macrophage activation thatcan be exploited therapeutically as it will be demonstrated elsewhere inthis application.

[0321] 2-TH acid is a potent inhibitor of cancerous cell growth. 2-TAacid, a thiofuran derivative, metal ion chelator, shows an effect on thegrowth and viability of normal and cancerous cells in tissue culture.Examples presented here show that 2-TH acid and derivatives have potentanti-viral and anti-cancer activity in vitro. Moreover, 2-TH acid may beuseful in the treatment of tumors in vivo without substantially damagingnormal cells. Furthermore, it has been shown that 2-TH acid andderivatives can inhibit a panel of 60 different cancer cell lines in anNIH screening assay.

[0322] One critical property of the novel compounds of this inventionderivatives of 2-TH acid is that the substitution at position 3 or 4with a 1 to 15 carbon saturated fatty acid group increases cellularpenetrability of this agent with respect to similar agents not havingthis lipid soluble residue. Inside the cells they works as specific TMAchelating agents. Another advantage is that inside the cell they canwork at picomolar or nanomolar concentrations, depending upon the agentused. In general, the chelating agents currently available workintracellularly at mM concentrations (Fernandez-Pol, 2001).

[0323] The 4-butyl-2-TH acid is the 4-butyl derivative of 2-TH acid. Itsstructure is shown in FIG. 1. It is clear that 4-B-2-TH acid by theactivity of the butyl group, or pentyl group as it will be shown later,penetrates the cell much more efficiently than 2-TH acid and dependingupon the characteristics of its derivatives will attack multipleintracellular target metalloenzyme systems and target ZFPs.

[0324] While some of the less specific agents of this invention willattack simultaneously multiple cellular targets, other agents of thisinvention, will be highly specific and they will attack only onespecific target metalloenzyme or target ZFP because of thecharacteristics of its molecular design and in particular thecharacteristics of their lipophilic side chains (FIG. 14). The threemost cytotoxic and specific chelators of this invention have an ID₅₀value of 0.1 uM, and were derived from 2-TH acid. Thus, this inventiondefines the 2-position and the 4-position of 2-TH acid as structuralcomponents that confer anti-neoplastic activity. This is attributed tothe high lipophilicity of the groups attached to position 4 and to thespecific chelating capacity of the groups attached to the carboxyl inposition 2. This relationship of lipothilicity to anti-proliferativeeffect was further documented by the observation that for a particularsubstitution at position 2 (e.g., hydrazide) the activity alwaysincreases as the lipophilicity of the parent compound increases (FIG.7). Significantly, the addition of a halogen at position 3 and/or 5increases the potency of the agent, possibly because of inhibition ofbiodegradation of the drug. Thus, in general, the specificity isconferred by both the lipophilic side chain and the chelating portion ofthe molecule, as demonstrated in several examples of this patentapplication.

EXAMPLE 1 Preparation of 4-butyl-3-5-Chloro-thiophenecarboxylic acidhydrazide

[0325] Many of the compounds described here are commercially available.Others have not been synthesized yet, although persons of ordinary skillin the art will know how to synthesize them. Thus, no attempt is madeherein to describe the synthesis of such compounds. The presentinvention is directed to the structure of matter for compounds that havenot been synthesize yet and to the use of both commercially availableand new heretofore unrecognized compounds that dissociate and bind zinc(or other transition metal ion) from metalloproteins, which therebyinactivate the pathogenic protein and subsequently the pathogenic lifeform, whether a virus, bacteria, fungus, parasite, or cancer cells.

[0326] The chemistry and synthesis of one of the analogues of 2-TH acidthat is of great interest for this invention can be described inexemplary form as shown in FIG. 2. This analogue denoted4-butyl-3-chloro-2-thiophenecarboxylic acid hydrazide can be 7-10 timesmore active than 2-thiophenecarboxylic acid against cancer cells intissue culture and against virally infected cells. Alternatively, theintermediary compounds shown in FIG. 2 can be derivatized to the2-hydrazone, and 2-thiosemicarbazone by means that are well known in theart. Protection of specific groups in the thiophene ring forhalogenation is well known in the art. See Protective Groups in OrganicChemistry, J. F. W. McOmie, Plenum Publishing Co., Ltd., 1973. Exemplarysources for the compounds that can be used as starting materials are asfollows: Sigma-Aldrich, St Louis Mo., USA and Fluka Chemical Co,Germany.

[0327] Other compounds of particular importance for the subjectinvention, derived from the formulas defined immediately above and shownin FIG. 2, have the following formulas:4-pentyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide,4-pentyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone and4-pentyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. orderivatives thereof.

[0328] In addition, the present invention provides methods for screeningthe compounds delineated above which have the appropriate electron donorand chelating characteristics which will be suitable for pharmacologicaluse.

EXAMPLE 2 Cellular Penetration of the Drugs Increases Cytotoxicity

[0329] The inventors have also recognized the importance of cellularpenetrability for the therapeutic efficiency of these agents in specialcircumstances such as penetration in anthrax-infected macrophages and ininfectious diseases of the brain. For that purpose, some of thecompounds of this invention were designed with special hydrophobicqualities.

[0330] As shown in FIG. 7, cytotoxicity of eight novel moleculescorrelated significantly with low water solubility. The increasehydrophobicity results in faster cellular penetrability of the drugswhich correlated with higher cytotoxicity. The molecules were morecytotoxic in the following order of increased hydrophobicity: (1)2-thiophenecarboxylic acid; (2) 2-thiophenecarboxylic acid hydrazide;(3) 4-butyl-2-thiophenecarboxylic acid hydrazide; (4)4-pentyl-2-thiophenecarboxylic acid hydrazide; (5)4-hexyl-2-thiophenecarboxylic acid hydrazide; (6)2-chloro-4-(trifluoromethyl)phenyl-2-furoic acid; (7)4-nitro-2-thiophenecarboxylic acid hydrazide; and (8)4,5-diphenyl-2-thiophenecarboxylic acid hydrazide.

[0331] Hydrophobicity may play a role in two cellular mechanisms of drugactivity: cellular penetration and access to specific subcellularcompartments. Therefore, better diffusion past the cellular membrane mayexplain the higher toxicity of the more lipid soluble molecules. Thedata confirms that increased hydrophobicity increases cytotoxicity asdemonstrated by the fact that the higher the water solubility, thehigher is the number of surviving cells.

[0332] In addition to the selection of compounds with high cellularpenetrability, methods for identifying compounds that have crossed thelipid barrier and can react with intracellular metalloproteins have beendeveloped in the last 20 years. These methods can be used to screen andidentify compounds based on their ability to react with peptidesequences selected from pathogenic metalloproteins. The reactions can becarried out in an aqueous or lipophilic environment, dependent upon thesolubility characteristics of the peptide and the drug being tested. Themethods of detecting the dissociation of the transition metal ion (TMI)ion or formation of ternary complexes (compound-TMI-protein) includeimmuno-blotting, Nuclear Magnetic Resonance (NMR), high pressure liquidchromatography (HPLC), detecting the release of radioactive TMI such asradioactive Zn2+, Cu2+ or Fe2+, detecting changes in protein mobility ingel shift assays, and capillary electrophoresis. Finally, assays tostudy the inhibition of binding of metalloproteins or metallopeptides toDNA or RNA by the chelating agents under study can be use to screen andidentify the compounds.

EXAMPLE 3 Molecular Structure of Systemic Compounds

[0333] Novel substituted derivatives of 2-thiophenecarboxylic (2-TH)acid and related compounds can be used systemically to treat viraldiseases, infections, cancer and other disorders as shown in Table 1.The novel substituted derivatives of 2-TH acid and related compoundsalso work by disrupting the binding of zinc atoms to zinc fingerproteins or by inactivating zinc in metalloenzymes, or other structuresheretofore unknown that depend upon the inclusion of zinc or othertransition metal ion (TMI), for stability, packaging or replication.Further, the novel substituted derivatives are stable and retain theirTMI chelating properties even when introduced systemically by injection,oral administration, inhalation or transdermal or other routes ofadministration.

[0334]FIG. 1 illustrates some of the novel derivatives of 2-TH acid forsystemic use. Computer modeling demonstrates that derivatives of 2-THacid can interact with zinc atoms and disrupt its binding to the targetmetalloprotein. Substitutions at positions 3,4 and 5 on the 2-TH acidhave the proper configuration to prevent interference with the zincfinger protein backbone. For example R1 or R2 can be a methyl, ethyl,propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiary butyl,pentyl, isopentyl, neopentyl or other hydrophobic or polar group.Further, substitutions with halogens such as chlorine, bromine, iodineand fluorine can result in highly stable and effective systemicallyactive agents. The systemic compounds can be prepared by methods wellknown in the art and include pharmacologically acceptable salts thereof.

[0335]FIG. 9 illustrates the binding of zinc in a zinc finger proteinand its disruption by the agents of this invention. Further, as shown inFIG. 14, the substitution at position 4 (R2) of the thiophene ring witha pentyl group can attach to amino acids on a hydrophobic pocket of themetalloenzyme, thus binding simultaneously to the zinc and thehydrophobic pocket, forming a ternary complex comprised of the protein,the zinc and the 2-TH acid derivative which inactivates themetalloenzyme. Therefore, the above-listed moieties that can besubstituted at various positions in the thiophene ring can result in a2-TH acid derivative that not only is more stable for systemicadministration, but also has even greater affinity, specificity, andselectivity for and binding potential with specific metalloproteins ormetalloenzymes.

[0336] It will be appreciated that substitution at the 3, 4 and 5positions can be made with single amino acids or peptides of 2 to 21amino acids containing either basic or acid amino acids in differentproportions. The substituted 2-TF acid will have an increased molecularweight and a substantially increased half-life in the blood. Further,the amphipathic nature of the peptide residues would allow a moreeffective penetration in abnormal cells, viruses, bacteria, parasitesand other microorganism.

[0337] The systemic compounds can be administered to animal and humansubjects orally, parenterally, inhalation, transdermally, rectally, orother pharmaceutically acceptable method of obtaining therapeutic levelsin the blood. In general, a pharmacologically effective daily dose ofthese derivatives could range from 0.01 mg/kg body weight to about 30mg/kg body weight per day. A pharmaceutically active preparation of 2-THacid or derivative in a concentration of 1% to 99% in a daily range of10 to 10000 mg, can be used for the treatment of various diseasesdescribed in the specific examples. It will be appreciated that 2-THacid and derivatives can be employed in topical preparations, inaddition to the systemic uses.

Wide-Spectrum Antiviral Activity of 2-Thiophenecarboxylic Acid andDerivatives

[0338] The chelating agents of the present invention can also be used tocontrol viral diseases. It has been documented that zinc and iron arerequired transition metals in certain proteins and enzymes essential forviral structure or function. For example, the replication of certainviruses such as the Hepatitis C virus is dependent on zincmetalloproteinases. Propagation of the AIDS virus is dependent on Zn2+requiring proteins such as the p7 zinc finger nucleocapsid protein. Theadministration of specific chelating agents of the instant invention canprevent unwanted formation of viral metalloproteins and metalloenzymes.The 2-TH acid derivatives can be highly efficient by targeting specificamino acid regions of the zinc finger or other metalloproteins ormetalloenzymes of viruses. These chemical entities that function in thesame manner as 2-TF acid and derivatives are intended to be encompassedby the instant invention. The chelating agent can be administered orallyor parenterally in doses described elsewhere in this application.Representative viruses with metalloproteins that can be targeted withthe agents of this invention are shown in Table 2. TABLE 2 Examples OfFamilies Of Viruses Using Zinc Finger Proteins Or Transition MetalIon-Dependent Enzymes For Replication And/Or Virulence that can betargeted with the agents of this invention Location and general ProteinFunction and Specific Families, Virus protein and Mr CharacteristicsProperties Reoviridae Lambda-1, 140 Kd Inner capsid Zinc finger proteinBinds dsDNA Rho-3, 41 Kd Outer capsid Zinc finger protein Binds dsRNARotaviridae NSP1, 53 Kd Non-structural Zinc finger protein RNA bindingRetroviridae np7 (AIDS) Nucleocapsid Two Zinc finger domains 55 aminoacids RNA binding Required for inclusion of RNA in virions ArenaviridaeMPS-1; 84 amino acids Regulatory protein One zinc finger domainPapillomavirinae E6 Regulatory protein Zinc finger protein Transformingprotein of HPVs Continuous cell proliferation Targets degradation of p53E7 Regulatory protein Zinc finger protein Transforming protein of HPVsContinuous cell proliferation Binds to the retinoblastoma protein, RbPoxviridae Ribonucletide Fe-dependent Synthesis of DNA Reductase EnzymePrecursors Flaviviridae (Hepatitis C) NS2(+NS3) Zn-dependent enzymeZn-metalloproteinase Herpesviridae- HSV-1: ICPO protein Regulatoryprotein Zinc finger DNA-binding Trans-activation HSV-2: MDBP proteinRegulatory protein Zinc finger protein ssDNA-binding DNA replicationICP6: Ribonucleotide Fe-dependent Synthesis of DNA precursors ReductaseEnzyme Orthomyxoviridae Influenza viruses M1 protein Structural proteinOne zinc-finger domain

EXAMPLE 1 2-thiophenecarboxylic (2-TH) Acid Inhibits the Zinc DependentBinding of Recombinant MPS-1/S27 Ribosomal Protein to DNA or RNA

[0339] MPS-1/S27 is a ribosomal protein involved in ribotoxic responsesto cellular damage, carcinogenesis and responses to viral infection(Fernandez-Pol, 2001). It is also induced-as a biological response tobacteria, fungus and parasites. In parasites such as Toxoplasma gondiithe parasitic encoded form of MPS-1/S27 is highly elevated and activeduring all the parasitic replication cycle. It is also active inmacrophages digesting cells such as those found in melanomas(Fernandez-Pol, 2001). MPS-1/S27 is also involved in biological responsemodulation by the chelating agents agents of this invention.

[0340] MPS-1 is described in detail in one of the inventor's U.S. Pat.No. Re: 35,585 (U.S. Pat. No. 5,343,041). MPS-1 has one zinc fingerdomain of the type CCCC (FIG. 8). By molecular modeling, the inventorshave determined that 2-TH acid and derivatives interact with the zincbound to the CCCC domain and removed Zn2+ from MPS-1. These dataindicate that 2-TH acid and derivatives should remove zinc and disrupt,denature or inhibit various types of zinc finger proteins ormetalloenzymes, whether known or heretofore undiscovered, includingviral proteins such as nucleocapsid p7 proteins, as will be explained inthe next example.

EXAMPLE 2 Identification of HIV-1 Nucleocapsid Protein p7 as a Targetfor 2-TH Acid and Derivatives

[0341] Research on the structure and function of ZFP, performed bynumerous investigators have identified the critical role that ZFP playin the uncontrolled proliferation of virally, chemically, and radiationtransformed cells in culture. ZFP are essential for DNA/RNA replication.By inhibiting ZFP in transformed cells, the uncontrolled proliferationof these cells is blocked. In addition, ZFP are required for packagingof viral genetic material into new virions (Fernandez-Pol, 2001).

[0342] The p7 protein of the HIV-1 virus contains two zinc fingers thatare essential for the recognition and packaging of viral RNA. Severalinvestigators have characterized the high affinity binding of HIV-1nucleocapsid protein (p7) to the HIV 5′LTR of viral RNA. The resultsshowed that p7 protein binds with high affinity (Kd's in the lownanomolar range) to short repeats of dTG's in RNA. Thus, drugs thatprevent the interaction of p7 protein with nuclei acids are of greattherapeutic interest.

[0343] As described above, it has been found that the p7 protein of theHIV-1 virus is required for correct assembly of newly formed virusparticles during the viral life cycle. In one embodiment of theinvention, the inventors have targeted p7 for drug therapy with 2-THacid and derivatives. Utilizing computer models, the inventors havediscovered the activity of 2-TH acid and derivatives in disrupting zincfinger nucleoproteins of retroviruses. FIG. 9 illustrates the effects of2-TH acid and derivatives on one of the zinc fingers of a retroviralprotein such as the p7 protein. Furthermore, FIG. 9 illustrates that thedisruption of one of the zinc finger binding domain in retroviralproteins caused by 2-TF acid and derivatives results in the ejection ofZn2+ and subsequent denaturing of the protein. Derivatives of 2-TF acidare zinc finger disrupting agents that act by specifically attacking oneor both of the two zinc finger domains of the retrovirus nucleocapsid p7protein.

[0344] Computer simulation has also shown that 2-TH acid and derivativesinduce an overall decrease in the number of complete viral particlesthat bud off and exit the cells to infect other cells. The HIV-1 andHIV-2 zinc finger p7 proteins are highly conserved in the zinc fingerdomains. It has been determined that the zinc finger domains are highlyconserved in the majority of retroviruses. Furthermore, mutations in thezinc fingers of the HIV-1 virus p7 protein produce a non-infectious UV-1viral particle. It has been determined that the zinc finger domains ofHIV viruses are essential for nucleic acid binding. Thus, p7 resistantmutants are unlikely to occur. Therefore, the 2-TF acid and derivativescan be used for prevention of retroviral diseases by chemically inducinga non-infectious viral particle and/or preventing the exit of a completeinfectious virus from the cells.

EXAMPLE 3 Inhibition of Herpes Virus Ribonucleotide Reductase (RR) byFuroic Acid and 2-TH Acid and Derivatives

[0345] The antivirals of this invention such as furoic acid and 2-THacid and derivatives can have a significant impact on the management ofherpes virus infections. The use of agents such as acyclovir,ganciclovir and foscarnet have resulted in an increase emergence ofdrug-resistant herpes virus strains. The use of the new classes ofanti-herpes virus compounds of this invention with novel mechanisms ofviral inhibition is important to prevent the emergence of viral mutants.Furoic acid, 2-TH acid and derivatives can simultaneously inhibit Herpesvirus RR, an iron requiring enzyme, and viral zinc finger proteins, thusreducing the possibility of emergence of Herpes virus mutant strains. Apharmaceutically acceptable concentration of furoic acid, 2-TH acid or aderivative in a concentration of 1% to 99% could be administered in adaily range of 100 to 5000 mg to inhibit replication of herpes viruses.Of course, these preparations can be combined with other anti-viralagents with different mechanisms of action to increase therapeuticefficiency.

EXAMPLE 4 Inhibition of Papilloma Virus Replication by the Action of2-Thiophenecarboxylic Acids

[0346] Only two viral ZFP of HPV are consistently expressed andintegrated in keratinocytes, the E6 and E7 zinc finger proteins. Theyare responsible for continuous cell proliferation. The E6 and E7proteins regulate cell proliferation by interfering with p53 and pRb,respectively. The cell cycle is altered at the G1/S interphase. Thus,one should be able to eliminate HPVs by using 2-TH acid and derivativesbecause the E6 and E7 HPV proteins are critical zinc finger proteinsrequired for viral replication. When replication of virus is arrested,apoptosis of virally-infected cells must occur. Thus, one may be able toalter the epidemiology of carcinoma of the uterine cervix by using 2-THacid or derivatives as chemopreventive agents.

[0347] 2-TH acid and analogues act by chelating metal ions. In the caseof inhibition of viral replication by 2-TH acid, the ion involved iszinc, which is essential to maintain the active structure of zinc fingerproteins such as E6 and E7 proteins of the human papilloma viruses whichare essential for viral replication.

[0348] Since HPV induces cell proliferation, the mechanism of action of2-TH acid may also be cellular, as that described for other virallytransformed cells. One possibility is that inhibition of growth by 2-THacid in HPV transformed cells can be explained by an effect on cellularRNA polymerase, a known metalloenzyme. It can also inhibit ribosomalprotein synthesis, since many ribosomal proteins are zinc fingerproteins (Fernandez-Pol, 2001). Thus, 2-TH acid and derivatives may acton numerous target sites such as viral, nuclear or cytoplasmicmetalloproteins. Furthermore, 2-TH acid and derivatives can act asbiological response modifiers, suggesting that when 2-TH acid andderivatives distorts the configuration of HPV zinc finger proteins, theymay become immunogenic when new antigenic sites are exposed. Thus, theagents of this invention can be used to stimulate a cellular immuneresponse against viruses, primary tumors and parasites.

EXAMPLE 5 Inhibition of Zinc Dependent Metalloproteinases of Hepatitis CVirus by 2-TH Acid and Derivatives

[0349] The hepatitis C family of viruses are dependent upon zincmetalloproteinases such as the NS2(+NS3) for replication of the virus(Table 2). 2-TH acid or suitable analogs, can be administered orally orparenterally to patients exposed or infected with Hepatitis C virus.This specific agents will bind the metal in the zinc dependentmetalloproteinase rendering it inactive and thereby controlling thedisease. Furthermore, the oral administration of the specific metalchelator in combination with other anti-viral agents may result in theelimination of the virus from the cells. A pharmaceutically acceptableconcentration of 2-TH acid or a derivative in a concentration of 1% to99% could be administered in a daily range of 100 mg to 5000 mg for thisantiviral treatment.

EXAMPLE 6 Inhibition of the AIDS Virus by a Metallo-Organic Complexes of2-Thiophenecarboxylic Acid and Derivatives

[0350] The active site of the HIV-1 protease contains a catalytic watermolecule between the two catalytic residues Asp25 and Asp125. Using databases and molecular modeling we observed that metallo-organic complexescan be form by using Cu (II)-2-TH acid and derivatives which fit intothe active site of the enzyme. We calculated that this competitiveinhibitor of the viral protease can work at the uM range. Furthermore,by adding a carboxyl in position 5 of the thiophene ring a bidentatecooper (1) chelator can be created. The bidentate copper (II) chelatorscan dock into the active site of the viral protease and inhibit itsactivity at the low uM range (5 to 0.1 uM).

EXAMPLE 7 The Microglia-Associated HIV Virus in Neurological Diseasescan be Inhibited by 4-butyl-2-thiophenecarboxylic Acid Hydrazide

[0351] HIV-1 is a neurotropic virus. The overwhelming majority of cellsinfected with HIV-1 in the central nervous system aremicroglia/macrophages. Microglia/macrophage infection leads to immunedeficiency as well as the production and release of cytotoxic moleculessuch as cytokines which contributes to the progression of the disease.Due to the capacity of the agents of this invention to cross the bloodbrain barrier, to penetrate and activate macrophages/microglia, and todisrupt HIV-1 viral zinc finger proteins, these agents can be used totreat this neurological viral condition.

EXAMPLE 8 Treatment of Chickenpox

[0352] Treatment of chickenpox can be accomplished by topical treatmentwith furoic acid or 2-TH acid when the rash is in the early stages. A 5%to 10% solution of the antiviral compound can be applied to the lesionsof a patient with chickenpox. Since herpes virus replication will beinhibited, it is expected that the lesions will not erupt into blistersand that the treated areas will not itch.

EXAMPLE 9 Treatment of Smallpox and Biological Warfare

[0353] The poxviruses are a family of large, enveloped DNA viruses. Themost notorious poxvirus is variola, the causative agent of smallpox.Smallpox was important because of the morbidity and mortality cause bythis virus. Despite the eradication of naturally occurring smallpox andthe availability of a vaccine, the potential for weaponization ofvariola virus continues to present a universal threat. The aerosolinfectivity of the virus, the ease of large-scale production, and anaïve human population remarks the importance of smallpox virus as aweapon. Vaccinia vaccination remains the preeminent countermeasure forsmallpox but its application and development of immunity is slow andthus it is not appropriate to counteract bioterrorism in all its forms.The drug N-methylisatin-p-thiosemicarbazone (Methisazone) possess someefficacy in post-exposure prophylaxis but is shows significant toxicity.An opportunity exist to correct and counteract this situation bydeveloping new, effective, low toxicity wide-spectrum antiviral agents.This invention contributes new wide-spectrum anti-viral agents suitablefor the purposes of prophylaxis and treatment of smallpox infections.

[0354] A large number of virus-encoded enzymes and factors are packagedin the smallpox virus particle. The RNA polymerase (a zinc requiringenzyme); the superoxide dismutase (a Cu/Mn/Zn requiring enzyme); andribonucleotide reductase (an Fe2+ requiring enzyme which is inhibited byhydroxyurea), as well as viral and cellular zinc finger proteins such asMPS-1/S27 ribosomal proteins are involved in critical early functions ofthe smallpox virus. Thus, the smallpox virus is dependent upon viral andcellular metalloproteins and metalloenzymes for replication of thevirus.

[0355] 2-Thiophenecarboxylic (2-TH) acid hydrazide, hydrazone,thiosemicarbazone, or suitable analogs, can be administered orally orparenterally to patients exposed or infected with smallpox virus. Thesespecific agents will bind the metal in the metalloenzyme and/ormetalloprotein rendering it inactive and thereby controlling thedisease. Furthermore, the oral administration of the specific metalchelator in combination with other anti-viral agents such as Methisazonemay result in the elimination of the virus from the cells. Apharmaceutically acceptable concentration of 2-TH acid or a derivativein a concentration of 1% to 99% could be administered in a daily rangeof 100 mg to 5000 mg for this antiviral treatment.

Cancer and Metastatic Disease 2-Thiophenecarboxylic Acid and Derivativesare Potent Inhibitors of Cancerous Cell Growth

[0356] The chelating agents of the present invention can also be used tocontrol cancer and metastatic disease. The administration of thespecific chelating agents of the instant invention can be used aseffective chemotherapeutic agents. The chelating agent can beadministered orally or parenterally in doses described elsewhere in thisapplication.

[0357] 2-thiophenecarboxylic (2-TH) acid, is a metal chelating compound,which inhibits the growth of numerous cultured normal and transformedmammalian cells. It also is shown that 2-TH acid can arrest prokaryoteand eukaryote cell growth by inhibiting Zn and Fe-requiring enzymes. Inaddition to its chelating ability, 2-TH acid has a number of biologicproperties such as macrophage activation.

[0358] 2-TH acid is a potent inhibitor of cancerous cell growth. 2-THacid, a thiofuran derivative, metal ion chelator, shows an effect on thegrowth and viability of normal and cancerous cells in tissue culture.Examples presented here show that 2-TH acid has potent anti-canceractivity in vitro. Moreover, 2-TH acid and derivatives can be useful inthe treatment of tumors in vivo without substantially damaging livingnormal cells.

[0359] 4-butyl-2-Thiophenecarboxylic acid hydrazide (4-B-2TAH) is aderivative of 2-TCA. Its structure is shown in FIG. 1. 4-B-2TAH wasrecognized to have potent anticancer activity in vitro. The propertiesof 4-B-2TAH can be summarized as follows: Undoubtedly the drug interactswith various zinc finger proteins and transition metal ion-requiringenzyme systems. 4-B-2-TAH is noted to be an inhibitor of a wide varietyof seemingly unrelated enzyme systems. These include Zn-finger proteinsand Zn-dependent metalloenzymes. Fe- and Cu-requiring enzyme systems arealso effected by 4-B-2-TAH. These enzymatic systems are important ingrowth control mechanisms. It is clear that 4-B-TAH, by virtue of itsbutyl group penetrates the cell interior much more easily than 2-THacid, and works as a Zn/Cu/Fe chelating agent.

[0360] Important, 2-TH acid and analogues, in particular the4-butyl-2-thiophenecarboxylic acid hydrazide (or hydrazone, orthiosemicarbazone) possess in vitro anti-tumor activity that is muchgreater and specific than currently available chelators. Furthermore,some of the compounds of this invention can act intracellularly atpicomolar concentration in transition metal ion containing enzymes, suchas zinc-dependent metalloproteinases. Thus, the agents presented in thisinvention can penetrate cells, reached the Zn-metalloprotein which ispresent at low intracellular concentrations (uM) and inactivate it.

[0361] From our studies with 2-TH acid analogues, we have identified themoieties shown in FIG. 1 and FIG. 2, as structural components whichinfer anti-neoplastic activity. The result indicate that theanti-proliferative activity of 2-TH acid and derivatives is due to theirability to form a metal ion complex.

[0362] It will be also appreciated that by understanding thestructure-activity relationships of these ligands, the compounds of theinstant invention can be made target specific and have dose-responserelationships for systemic use that are sigmoidal and thus they have awide range of therapeutic concentrations (FIG. 1, FIG. 4, FIG. 14).

[0363] Examples of the specific effects of metal chelating agents,including furoic acid, and 2-thiophenecarboxylic acid and derivatives,as well as the practical applications of these agents in medicine willnow be described:

EXAMPLE 1 Effects of 2-Furoic Acid on Growth of CHO Cancer Cells

[0364] Cells were plated at 1.5×10⁵ cells/60-mm dish; 24 hours later,the medium was removed, and new media with or without 0.05 to 10 mMfinal concentrations of 2-Furoic acid were added. Cell counts weredetermined at 24, 48 and 72 h after the addition of 2-Furoic acid; eachpoint is the average of triplicate measurements from 3 cultures. Furan,an analog of 2-Furoic acid, was simultaneously tested at 0.05 to 10 mMunder identical culture conditions (FIG. 4).

[0365] The results show that the growth of CHO cells was inhibited in adose-dependent fashion by 3 to 10 mM 2-Furoic acid within 24 hours. Thecells showed no significant toxic effects for up to 72 hours aftertreatment. At 72 h the growth inhibition induced by 3 and 10 mM 2-Furoicacid were about 20% and 40%, respectively. Furan, a structurally relatedsubstance had a similar dose-dependent effect on cell growth inhibitionat 24, 48 and 72 h (FIG. 4).

EXAMPLE 2 Effects of 2-Thiophenecarboxylic Acid Hydrazide on Growth ofCHO Cancer Cells

[0366] Cells were plated at 1.5×10⁵ cells/60-mm dish; 24 hours later,the medium was removed, and new media with or without 0.05 to 10 mMconcentrations of 2-thiophenecarboxylic acid hydrazide (2-TCAH) wereadded to the media. Cell counts were determined at 24, 48 and 72 h afterthe addition of 2-TCAH; each point is the average of triplicatemeasurements from 3 cultures. Thiophene (Thiofuran), an analog of2-TCAH, was simultaneously tested at 0.05 to 10 mM under identicalculture conditions (FIG. 4).

[0367] The results show that the growth of CHO cells was inhibited in adose-dependent fashion by 1 to 10 mM 2-TCAH within 24 hours. The cellsshowed significant toxic effects at 48 to 72 hours after treatment (FIG.6B). At 72 h the inhibition induced by 1 and 10 mM 2-TCAH were about 65%and 90%, respectively. Thiophene, a structurally related substance hadno significant effect on cell growth at 72 h (FIG. 4).

EXAMPLE 3 Effects of 3,4,5-Trichloro-2-Furoic Acid on the Growth ofCancer CHO Cells

[0368] Cells were plated at 1.5×10⁵ cells/60-mm dish; 24 hours later,the medium was removed, and new media with or without 0.1, 1 and 3 mMfinal concentrations of 3,4,5-Trichloro-2-Furoic acid (3,4,5-TFA) wereadded. Cell counts were determined at 24, 48 and 72 h after the additionof 3,4,5-TFA; each point is the average of triplicate measurements from3 cultures.

[0369] The results show that the growth of CHO cells was stronglyinhibited by 1 mM and 3 mM 3,4,5-TFA within 12 h of addition of thesubstance (FIG. 5; FIG. 6C). At 24 h, 48 h and 72 h the growthinhibition induced by 1 and 3 mM 3,4,5-TFA was greater than 80% and 99%,respectively (FIG. 5; FIG. 6C).

[0370] After 12 to 48 hours of exposure to 1 and 3 mM 3,4,5-TFA, CHOcells acquired a flattened morphology, they began to look granular, nomitosis were observed, and some began to float in the medium. Withlonger exposure (48-72 hours) cytotoxicity and cell death was observedin greater than 99% of CHO cells treated with 3 mM 3,4,5-TFA (FIG. 6C).

EXAMPLE 4 Effects of 3,4,5-Trichloro-2-Thiophenecarboxylic Acid on theGrowth of Cancer CHO Cells

[0371] Cells were plated at 1.5×10⁵ cells/60-mm dish; 24 hours later,the medium was removed, and new media with or without 0.1, 1 and 3 mMfinal concentrations of 3,4,5-Trichloro-2-Thiophenecarboxylic Acid(3,4,5-TFA) were added. Cell counts were determined at 24, 48 and 72 hafter the addition of 3,4,5-TTA; each point is the average of triplicatemeasurements from 3 cultures.

[0372] The results show that the growth of CHO cells was stronglyinhibited by 1 mM and 3 mM 3,4,5-TTA within 6 h of addition of thesubstance. At 24 h, 48 h, and 72 h, the growth inhibition induced by 1mM and 3 mM 3,4,5-TTA was greater than 98% and 100%, respectively (FIG.5).

[0373] After 6 to 12 hours of exposure to 1 and 3 mM 3,4,5-TTA, CHOcells acquired a flattened morphology, they began to look granular, nomitosis were observed, and some began to float in the medium. Withlonger exposure (24-72 hours) cytotoxicity and cell death was observedin greater than 99.9% of CHO cells treated with 1 mM or 3 mM 3,4,5-TTA(FIG. 6C).

EXAMPLE 5 Effects of 3,4,5-Trichloro-2-Thiophenecarboxylic AcidHydrazide on the Growth and Viability of Cancer CHO Cells

[0374] The growth of CHO cells was strongly inhibited by 500 uM3,4,5-Trichloro-2-Thiophenecarboxylic Acid Hydrazine (3,4,5-TTAH). After12 to 24 hours of exposure to 500 uM 3,4,5-TTAH, CHO cells acquired aflattened morphology, they began to look granular, no mitosis wereobserved, and numerous cells began to float into the medium. With longerexposure (24-72 hours) cytotoxicity and cell death was observed ingreater than 95% of CHO cells. The detached cells showed conspicuouscytotoxic effects and most of them (>99.9%) were destroyed by the agent,as determined at 72 h using the trypan blue dye exclusion test.

EXAMPLE 6 Effects of 4-butyl-2-Thiophenecarboxylic Acid Hydrazide on theGrowth and Viability of Adenocarcinoma Cells

[0375] 4-butyl-2-Thiophenecarboxylic Acid Hydrazide (4-B-2-TAH; 50 μM)can inhibit human breast adenocarcinoma MDA-468 cell growth. After 12hours of treatment with 50 μM 4-B-2-TAH, there was no further increasein cell number. When treated with 50 μM 4-B-2TAH, the majority of theMDA-468 cells became granular, showed pronounced cytotoxic effects, manywere destroyed and subsequently detached from the culture dish. Thesefloating cells were not viable. Within 48 h of treatment there wasgreater than 90% decrease in cell number. Thus, breast adenocarcinomaMDA-468 cells are extremely sensitive to the cytotoxic actions of4-B-2-TAH. Thus, 4-B-2TAH can be effective to reduce and control growthof this common type of human malignancy and possibly other types ofhuman adenocarcinoma such as colon or lung adenocarcinoma.

EXAMPLE 7 Combined effects of Furoic Acid, 2-Thiophenecarboxylic Acid,or Derivatives Thereof with Standard Chemotherapeutic Agents

[0376] Other chemotherapeutic agents such as 5-fluorouracil,vinblastine, taxol and levamisole, in the case of breast or colonadenocarcinoma, may be utilized in conjunction with the agents of thisinvention to enhance the effectiveness of cancer therapy. For example,cancer cell death and biological alterations induced by4-butyl-3,5-difluor-2-Thiophenecarboxylic Acid-Thiosemicarbazone, anagent that molecular modeling and the study of the structure-activityrelationships indicates that it has potent anti-neoplastic activity, maybe enhanced by using agents from the group consisting of anti-cancerantibodies, radioactive isotopes, and chemotherapeutic agents.

[0377] The method of using furoic acid or thiophenecarboxylic acids andderivatives thereof, topically to treat a variety of viral andspontaneous proliferative diseases in human and animal subjects, as willbe described in detail below, can be used in combination with cytotoxicagents selected from the group consisting of chemotherapuetic agents,antibodies, and cytokines (e.g. Interferons), for enhanced activity.

Dermatological Use of the Agents of This Invention

[0378] Therapy to correct some of the pathological conditions of theskin can be accomplished by the agents of this invention which may bedelivered topically, systemically, or intralesionally. Topical therapyis a convenient method of treatment but its efficacy depends onovercoming the barrier function of the skin, primarily that presented bythe stratum corneum. The agents of this invention are important astopical and systemic therapeutic agents for skin diseases. Orally, thisagents can be used to treat numerous dermatological diseases thatinclude abnormal proliferation or infections. Potent and efficaciousderivatives of 2-TH acid can be used for the treatment of acne,psoriasis, and other skin diseases. Modifications of such molecules mayresult in topical agents that can be used for their anti-carcinogenicand antiaging effects. The following examples delineate some of the skindiseases that can be treated by the agents of this invention.

EXAMPLE 1

[0379] Treatment of Psoriasis

[0380] Psoriasis is characterized by the pathologically rapid epidermalproliferation induced by an immune-mediated dermal inflammation. Furoicacid or 2-TH acid and derivatives can be used to inhibit cellproliferation and prevent the inflammatory condition of psoriasis. Theprimary therapeutic mechanism of these agents centers in both inhibitionof hyperproliferation and reduction of epidermal inflammation. A usualdose for topical therapy is 5% to 10% of furoic acid or 2-TH acid in anabsorption base applied two times daily. Systemic administration can beused in the doses described elsewhere.

EXAMPLE 2 Treatment of Acne

[0381] Acne is a common skin disorder through the world, affecting about7% of the population between 12 and 24 years old. Acne is a disease ofthe pilosebaceous unit. The pathogenesis of acne includeshyperproduction of sebum, growth of Propionobacterium acnes, andinflammation. Furoic acid or 2-TH acid and derivatives can decreasesebum production, inhibit P. acnes grow, and reduce inflammation,leading to the control of acne. The lesions can be treated with anaqueous solution or an absorption base containing approximately 5% to20% furoic acid or 2-TH acid or derivatives thereof. The pharmacologicalagents can also be used systemically and intralesional in formulationsdescribed elsewhere in this application.

EXAMPLE 3 Treatment of Skin Infections

[0382] Infections of the skin may be bacterial, viral fungal andparasitic. Topical application of the agents of this invention can beused for the control rosacea, impetigo and other skin infections. Viralinfections are numerous and include verrucae (human HPV), herpes simplex(HSV), condylomata acuminatum (HPV), molluscum contagiosum (poxvirus)and chicken pox (varicella) among the most common diseases of the skin.Few medications are available for viral diseases of the skin. Furoicacid or 2-TH acid and derivatives thereof can be used to control viraldiseases such as Herpes, HPV and molluscum contagiosum. They can also beused to control fungal and parasitic diseases. The lesions can betreated with an aqueous solution or an absorption base containingapproximately 5% to 20% furoic acid or 2-TH acid or derivatives thereof.When indicated, the pharmacological agents can also be used systemicallyand intralesional in formulations described elsewhere in thisapplication.

EXAMPLE 4 Treatment of Exposure to UV Radiation

[0383] The exposure of the skin to UV radiation such as sun exposureinduces molecular and cellular damage which results in a cellularinflammatory response that includes overproduction of heat shockproteins. The compounds and methods of the present invention can be usedto block the excessive molecular stress response caused by UV radiation.The compounds block excessive production of zinc finger proteinsinvolved in inflammation such as DnaJ proteins which are involved inheat shock responses. Formulations of 1% to 10% furoic acid or 2-TH acidand derivatives thereof in an adequate solution or absorption base canbe used to treat sunburn.

EXAMPLE 5 Treatment of Neoplasms of the Skin

[0384] The agents of this invention can be use for the treatment andprevention of neoplasm of the skin. The pharmaceutical properties anddoses of these agents are discussed elsewhere in this application.Actinic (or solar) keratoses are potentially serious cutaneous neoplasmsthat are due to chronic UV radiation exposure. Prevention and treatmentof Actinic keratoses can be accomplished by topical applications ofFuroic acid or 2-TH acid and derivatives thereof. Topical concentrationsof 1%,2% and 5% as creams or solutions may be applied twice a day or inan alternative acceptable regime.

EXAMPLE 6 Simultaneous Activation of Macrophages and Induction ofApoptosis in Melanoma Cells by 2-TH Acid: Macrophages in and Around theAreas of Metastatic Melanoma Phagocytize Apoptotic Melanoma Cells AfterTreatment with 2-TH Acid

[0385] We have previously observed that macrophages in melanomas arevery active, suggesting a direct role of macrophages in phagocytosis ofmelanoma cell debris following apoptosis, a common phenomenon. Incontrast, benign nevus rarely show macrophages or they are non-existent.We have found that 2-TH acid and derivatives can stimulate macrophagesto digest apoptotic melanoma cells. Apoptosis in melanoma cells isinduced by the agents of this invention. Thus, the agents of thisinvention can be used topically or systemically to stimulatephagocytosis of melanoma cells by macrophages in and around the areas ofmetastatic melanoma cells which are simultaneously induced to enter intoapoptosis by 2-TH acid and derivatives thereof. The lesions can betreated with an absorption base containing approximately 5% to 20% 2-THacid or derivatives thereof. When indicated, the pharmacological agentscan also be used systemically and intralesional in formulationsdescribed elsewhere in this application.

EXAMPLE 7 Treatment of Poison Ivy Contact Dermatitis

[0386] The Anacardiaceae are known for the toxic components of some ofthe members of this family, specially poison ivy (Toxicodendronradicans), poison oak, and poison sumac in North America. This plantproduces contact dermatitis due to the production of urushiol which is acomplex mixture of lipophilic immunogenic cellular protein-bindingcompounds. The antigenic component of poison ivy, oak and sumac iscalled urushiol. Poison ivy urushiol is mostly composed ofpentadec(en)yl catechols.

[0387] The antigens of allergic poison ivy contact dermatitis are lipidsoluble and bind to specific proteins of the skin Langherans cells (skinmacrophages). In the Langerhans cells, the lipophilic urushiol antigensare internalized by endocytosis with the antigen subsequently degradedto be presented to T-Lymphocytes. This universal mechanism of antigenprocessing also occurs with viral, bacterial, fungal, tumor, ortransplantation antigens as well as with urushiol. Subsequently, theT-lymphocytes recognize the antigens (urushiol) with the help of theantigen presenting cells (Langerhans cells) and the poison ivy reactionis initiated.

[0388] Poison Ivy can be alleviated by the use of the agents of thisinvention. The agents can be used to increase the protein-antigendegrading activity of macrophages (Langherans cells) and to inhibit theT-cell lymphocytes inflammatory response. The poison ivy, oak or sumaclesions can be treated with an absorption base containing approximately5% to 20% 2-TH acid or derivatives thereof. When necessary, thepharmacological agents of this invention can also be used systemicallyin formulations described elsewhere in this application.

Ocular Pharmacology

[0389] The antimicrobial agents of this invention can be used to treatocular diseases. The agents can be employed as antivirals for herpessimplex of the eye or as antibiotics for endophtahnitis. They may alsohave uses in fungal and parasitic diseases of the eye.

EXAMPLE 1 Treatment of Viral Keratitis

[0390] Viral keratitis, an infection of the cornea, is most commonlycaused by herpes simplex type I and varicella zoster viruses. Herpes IIand cytomegaloviruses can also cause keratitis. Topical antivirals ofthis invention can be use for the control of these conditions. Thetopical of intraocular ophthalmological preparation includes from 0.01%to 5% 2-TH acid or its substituted derivatives in an osmoticallyappropriate vehicle.

EXAMPLE 2 Treatment of Fibroblast Hyperproliferation in the Eye

[0391] Hyperproliferation of fibroblasts in the eye after cataractsurgery or implantation of artificial crystalline lens, may result inthe opacity of the lens. At present, 5-flurouracil (5-FU) and otherantiproliferative agents are instilled in the eye to control fibroblastproliferation. However, 5-FU is difficult to control and has untowardeffects. The chelating agents of this invention can be used to controlfibroblast hyperproliferation. An additional potential advantage ofthese agents are the simultaneous effects as inhibitors of angiogenesis,making 2-TH acid and derivatives suitable drugs after ophthalmicsurgery.

2-Thiophenecarboxylic Acid and Pharmacologically Acceptable DerivativesThereof in the Dosages Delineated Above can be Used to Prevent theFormation of Aberrant Iron and Copper-Finger Proteins Involved inCarcinogenesis and Aging

[0392] The chelating agents of the present invention can also be used aschemopreventive agents to control the formation of aberrantmetalloproteins involved in carcinogenesis and aging.

[0393] Transition metal ions at physiological concentrations, such asiron, cobalt, copper, etc., are essential elements for biologicalfunctions, however at higher levels they are toxic. This is particularlytrue for iron. Elevated levels of iron contribute to carcinogenesis inseveral ways: First, iron has the capacity to generate highly reactivefree radicals which damage DNA; and Second, there is an increased ironrequirement by rapidly proliferating transformed cells for DNAreplication (ribonucleotide reductase) and energy production bymitochondria (Fernandez-Pol, 2000, 2001).

[0394] Recent studies offer new insight into the mechanisms andpotential for damage to DNA by transition metals, particularly by ironand copper. These new insights result from the discovery thattranscriptional regulatory proteins that interact with DNA (DNA bindingproteins) which normally bind zinc (zinc finger domains) but which cansubstitute zinc by other transition metals present in the cell atabnormal concentrations may be involved in the degradation of DNAgenetic regulatory response elements leading to carcinogenesis andaging.

[0395] Heavy metal incorporation into zinc finger proteins (ZFP) may beimportant in metal-induced toxicity. An iron-substituted zinc finger maygenerate free radicals which damage DNA and potentially inducescarcinogenesis. The capability of iron to replace zinc in zinc finger,denoted the iron finger, was demonstrated in a series of experimentsboth in vivo and in vitro. Iron has the ability to substitute for zincin many ZFP. The iron finger in the presence of H₂O₂ and ascorbategenerates highly reactive free radicals (hydroxyl), producing areproducible cleavage pattern to the DNA of the respective responseelement. The close proximity of the zinc finger to DNA, as found bycomputer modeling, suggests that the iron-substituted zinc finger maygenerate free radicals while bound to genetic regulatory responseelements, leading to degradation of DNA and/or carcinogenesis(Fernandez-Pol, 2001). In summary, data at the molecular and clinicallevel support the notion that biologically essential heavy metals andfree radicals influence the aging process and induce carcinogenesis byinterfering with normal functions of regulatory ZFP.

[0396] Transition metal ions, particularly cupric ions and complexescontaining Cu2+⁺ and ferric ions and complexes containing Fe3+ candissociate and replace the zinc ion from the zinc finger of importantregulatory proteins. For example, zinc finger containing hormonereceptor proteins for testosterone, progesterone, etc, can replace zincby iron and may generate free radicals which damage DNA in specificregulatory regions and potentially induced carcinogenesis in prostate,uterus, etc, respectively. Thus, classical hormones can modulate ironfinger receptor proteins, suggesting that these hormones potentiate thedestructive actions of free radicals, mediated by abnormal iron fingerreceptor proteins, on regulatory regions of DNA.

[0397] The inventors have determined that it is feasible to maintainzinc finger proteins in an undamaged zinc-containing form by using acombination of specific chemopreventive agents such as specific ironchelators and radical scavengers that, respectively, interfere with theformation of both aberrant iron finger proteins and free radicals. Thus,2-TH acid and pharmacologically acceptable derivatives thereof in thedosages delineated above can be used to prevent the formation ofaberrant iron and copper-finger proteins involved in carcinogenesis andaging.

Metalloenzyme Targets

[0398] Matrix metalloproteinases, also called matrixins, are a family ofstructurally related Zn2+ enzymes that mediate the breakdown ofconnective tissue. Today more than 20 enzymes are known. The most commonbelong to the families of collagenases, gelatinases, and stromelysins.The substrates for these enzymes include collagens, elastin,proteoglycans, serpin, and gelatin. The functions of matrixmetalloproteinases include trophoblast invasion, mammary glandinvolution, and skeletal and limb development.

[0399] One important feature of the matrixins is that many of thesegenes are “inducible”. The effectors include growth factors, cytokines,chemical agents, viruses, etc. These enzymes are highly regulated byendogenous proteins inhibitors (e.g. alpha2-macroglobulin) and tissueinhibitors of metalloproteinases (TIMPs). Abnormal regulation occurs innumerous diseases such as invasive tumor growth and angiogenesis,rheumatoid arthritis, and in aneurysms.

[0400] All matrixins are synthesized as prepro-enzymes and secreted asinactive pro-MMPs in the majority of cases. The matrixins contain twozinc atoms, a catalytic zinc and a structural zinc. The catalytic zincis bound to 3 histidines within a conserved sequence. In inactiveenzymes the catalytic Zn is also bound to a cysteine SH group in anotherconserved sequence. The mechanism of action of matrixins includes thecoordination of the carbonyl carbon of the peptide bond to zinc whichresults in nucleophilic attack and subsequent peptide cleavage.

[0401] More specifically, the pro-peptide domain has a conserved uniquePRCG(V/N)PD sequence. The cysteine within this sequence (the “cysteineswitch”) binds the catalytic zinc to maintain the latency of pro-MMPs.The catalytic domain contains a highly conserved zinc binding motifHEXXHXXGXXH. The catalytic domains of matrixins have an additionalstructural zinc ion and 2 to 3 calcium ions, which are required forstability and expression of enzymatic activity.

[0402] It has been determined that the effective inhibitors of matrixinsof the instant invention must have the following groups: 1)onefunctional group capable of binding to the catalytic zinc such ascarboxylic acid, thiol, or hydroxamic acid; 2) have at least onefunctional group which can H-bond with the enzyme backbone; and 3) haveone or more side chains capable of favorable London interactions withthe enzyme active site.

[0403] Examples of the specific effects of metal chelating agents onmatrixins, including 2-thiophenecarboxylic acid and derivatives, as wellas the practical application of those agents will now be described:

EXAMPLE 1 Simultaneous Inhibition of Tumor Angiogenesis (VascularEndothelial Cell Proliferation) Cancer Cell Proliferation and Invasionby 2-Thiophenecarboxylic Acid and Analogues Inhibitors of Zinc-DependentMatrix Metalloproteinases (MMP)and Copper Requiring Enzymes

[0404] The chelating agents of the present invention can also be used tocontrol the initiation of neovascularization in various diseaseconditions. Neovascularization is dependent on zinc-requiring matrixmetalloproteases (Zn-MMPs). The administration of the specific chelatingagents of the instant invention can prevent unwanted angiogenesis. Thechelating agents can be administered orally or parenterally in dosesdescribed elsewhere in this application.

[0405] One group of proteolytic enzymes susceptible to the agents ofthis invention are the Zn-MMPs. Controlling Zn-MMP activity using thesynthetic small molecule inhibitors of this invention is an importantmechanism to stop several of the rate-limiting steps in this pathwayleading to angiogenesis and invasion. As shown in FIG. 10, inhibition ofangiogenesis can be accomplished by the simultaneous inhibition of thefollowing entities: 1) MMPs which are involved in proteolysis of theintracellular matrix, 2) cell migration; and 3) chemotaxis; and cellproliferation.

[0406] The growth of solid tumors depends on neovascularization,extensive cell proliferation, and local migration of cancer cells (FIG.10). Angiogenesis can be considered an invasive process in whichactivated vascular endotbelial cells proliferate, adhere toextracellular matrix molecules, and migrate. A similar sequence ofevents regulates cancer cell invasion. Zinc-dependent MMPs are involvedin both the angiogenic and the invasive process. Zn-MMPs degrade theextracellular matrix molecules and create a permissive environment forcell invasion and migration. Malignant tumors are characterized by anincrease activity of Zn-MMPs.

[0407] The 2-thiophenecarboxylic acid and derivatives can actsimultaneously to inhibit angiogenesis, cell proliferation (vascularendothelial and cancerous cell), and cellular migration (FIG. 10). Solidtumors are characterized by high proliferation rate, extensiveangiogenesis, aggressive local invasion, and metastasis, whicheventually make these tumors resistant to conventional treatment such assurgery, chemotherapy and radiotherapy.

[0408] Our data shows that 2-TH acid and derivatives can interfere withmalignant tumor development by both angiogenic dependent and independentmechanisms. More specifically, the 4-butyl-2-Thiophenecarboxilic acidhydrazide (4-B-2-TAH) was recognized by the inventors to have ananticancer and antiangiogenic activity in vivo.

[0409] The properties of 4-B-2-TAH can be summarized as follows: Thedrug interacts with various target metalloproteins such as Zn-MMPs (FIG.10). Furthermore, it has been shown that Cu2+ requiring enzyme systemsare essential for tumor angiogenesis. These Cu2+ metalloenzymes are alsoinhibited by 4-B-2-TAH (FIG. 10). This agent also affects zinc fingerproteins and ribonucleotide reductase that are important in growthcontrol mechanisms of both endothelial and cancer cells. It is clearthat 4-B-2-TAH not only inactivates Zn-MMP extracellularly, but byvirtue of its 4-butyl group penetrates the cell interior much moreeasily than 2-TH acid, and works as an specific intracellular Zn/Cu/Fechelating agent, inhibiting malignant tumor formation at multiple targetpoints (FIG. 10).

[0410] More specifically, 4-B-2-TAH at μM concentrations can covalentlyand coordinately bind to the catalytic zinc-containing site of theZn-MMPs, rendering the enzyme inactive. Furthermore, uM concentrationsof 4-B-2-TAH showed pronounced cytotoxic effects in many differentcancer cell lines in vitro which were destroyed by this agent and thuswere not viable. Therefore, 4-B-2-TAH can inhibit Zn-MMPs involved inangiogenesis and also can inhibit cancer cell growth in vitro of manyadenocarcinoma cells which are extremely sensitive to the cytotoxicactions of this agent. Thus, 4-B-2TAH can be effective to simultaneouslycontrol angiogenesis (inhibition of endothelial cell proliferation,Zn-MMPs, and Cu2+ metalloenzymes), cancer cell growth (disruption ofzinc finger proteins and inhibition of ribonucleotide reductase), andcell migration-inhibition of Zn-MMPs) in many common types of solidtumors such as breast, colon or lung adenocarcinoma.

EXAMPLE 2 Treatment of Pulmonary Anthrax by Blocking Lethal Factor, aHighly Specific Zinc-Dependent Metalloprotease that Contains aZinc-Dependent Catalytic Center

[0411] The chelating agents of the present invention can also be used tocontrol systemic infections produced by B. Anthracis spores in variousorgans, including cutaneous, intestinal, and the most deadly forms ofthis disease, meningitis and pulmonary anthrax in animals and humans.

[0412] The compounds of this invention can be used to effectivelyneutralize the Lethal Factor (LF) of B. Anthracis. The LF belongs to thefamily of zinc-dependent metalloproteases. LF contains a zinc-dependentcatalytic center which is essential for the proteolytic and pathogenicfunctions of this protein. As will be shown later, the zinc-dependentcatalytic center of LF can be covalently neutralized by the agents ofthis invention.

[0413] LF is a zinc-metalloenzyme target for the specific therapeuticagents of this invention such as 2-Thiophenecarboxylic acid hydrazide(2-THA), 4-Butyl-THA-hydrazide, 4-butyl-THA-hydrazone or4-butyl-TCA-thiosemicarbazone, and their derivatives thereof, that caninhibit the catalytic activity of LF by covalent binding to thezinc-dependent catalytic motif of LF. The 2-TH and derivatives thereofare powerful metalloprotease inhibitors that can block the toxic effectsof LF by covalent binding to the zinc-dependent catalytic domain of LFin vitro and in vivo, as determined by molecular and cellulartrafficking modeling.

[0414] It is the purpose of this example to demonstrate that some of thespecific compounds of this invention with special characteristics (e.g.specific covalent binding to the zinc-depend catalytic center) can beused to neutralize the LF toxin which conceivably could provide a modernlife-saving tool and protecting agent in an emergency situation in whichthe pulmonary or meningeal disease has progressed to a point whereantibiotic treatment is ineffective.

[0415] Anthrax is a significant agent of biological warfare andterrorism in the form of spores which when inhaled produce pulmonaryAnthrax. Although antibiotics can control certain forms of Anthrax,inhalation anthrax, which is produced by the spores of Bacillusanthracis is fatal in the majority of cases due to the late diagnosis ofthe disease making antibiotic treatment ineffective.

[0416] Research in the pathogenesis of anthrax identified two uniquevirulence factors of anthrax pathogenesis: poly-D-glutamic acid capsuleand a tripartite protein toxin.

[0417] The anthrax toxin has three components which act in concert:protective antigen (PA), oedema factor (EF), and lethal factor (LF). ThePA is a four-domain protein that binds a host cell-surface receptor;cleavage by a furin-like protease allows PA to form heptamers that bindthe toxic enzymes EF and LF. Subsequently, the complex is endocytized,inserted into the endosome membrane, followed by translocation of the EFand LF into the cytosol. The binary combination of PA and LF (lethaltoxin) is sufficient to induce rapid death in animals when givenintravenously.

[0418] Of significant importance for this invention is that certainmetalloproteases inhibitors can covalently bind to the Zn-dependentcatalytic center of LF, and they can block the toxic effects of LF invitro and in vivo, as determined by molecular and cellular modeling.Therefore, LF has been identified as a target for therapeutic agentsthat can inhibit its catalytic activity or block its association withPA.

[0419] PA possesses a high affinity binding site for which the LF andthe EF catalytic components compete. The PA63 complexed to the catalyticcomponent undergoes receptor mediated internalization and translocationinto the cytosol where the LF and EF are released to perform theirenzymatic activities. EF is an adenylate cyclase, calmodulin-dependent,and hence only functional in eukaryotic cells.

[0420] Lethal Factor (LF) is a protein (Mr=90,000) that is instrumentalin the pathogenesis of anthrax (FIG. 11). It is a highly specificmetalloprotease that cleaves proteins belonging to the family ofmitogen-activated protein kinase-kinase (MAPKK), leading to theinhibition of various signaling pathways. This protein is related to thezinc metalloprotease family and contains a zinc-dependent catalyticcenter. LF contains one or more Zn²⁺-binding amino acid motifs, one ofwhich is characteristic of the thermolysin family of zinc-dependentmetalloproteases. The MAPKK family of proteins are the only knowncellular substrates of LF. In tumors, the LF toxin inhibits tumor cellgrowth and angiogenesis, most likely by inhibiting the MAPKK-1 andMAPKK-2 pathways.

[0421] The binary combination of PA+LF is lethal to laboratory animalsand lysis cells of the monocyte/macrophage lineage. In pulmonaryanthrax, the LF leads to lysis of macrophages within 60-90 minutes byapoptosis. When the lethal toxin (PA+LF) is administered intravenouslyto rats, death is produced within 60-90 minutes and is characterized bysevere pulmonary edema. The lethal effects on macrophages in vitro hasbeen demonstrated to be calcium-dependent. Chelators of Ca²⁺ such asEGTA can protect macrophages from the lytic effect of the LF.

[0422] Antibiotics are not active against the spore forms of B.Anthracis. Although antibiotics are administered prophylactically tosubjects exposed to aerosolized spores, it may be considered prudent andadvantageous to administered an anti-LF agent which will neutralize thedeleterious effects of this potent toxin.

[0423] The anthrax toxin lethal factor binds multiple zinc atoms.Results from atomic adsorption spectroscopy indicate that LF containsapproximately three zinc atoms per toxin molecule. LF treated with EDTAand o-phenanthroline contained a similar number of zinc atoms,indicating that all three zinc atoms are tightly bound to the protein.LF contains the highly conserved zinc-binding consensus sequence, HexxH,that is present in all known zinc metalloproteases. In addition, lethalfactor contains an invented form of the motif, HxxDH, which may also beinvolved in zinc binding.

[0424] Molecular modeling studies showed that a zinc ion (Zn²⁺) iscoordinated tetrahedrally by a water molecule and three protein sidechains, in an arrangement resembling the thermolysin family. We built acomputer peptide model that shows that the compound4-butyl-THA-hydrazine can make covalent contact with the catalyticallyactive Zn²⁺, leading to a conformational change that neutralizes themetalloprotease activity of the LF. The surfaces on the adjacent regionof the Zn²⁺ binding site can also provide additional docking sites forderivatives of the 4-butyl-THA-hydrazine family that can be use astherapeutic agents that will block the activity of LF in vivo.

[0425] It will be appreciated by those skill in the art that theinventor has disclosed the best mode of the invention. Therefore, theforegoing specifications and accompanying drawings (FIG. 11) areintended to be illustrative of this example only.

[0426] Thus, suitable doses of the disclosed 4-butyl-THA-hydrazine andderivatives thereof, particularly those shown to penetrate macrophagesand to cross the blood-brain barrier (4-butyl-TFA-thiosemicarbazone),which can be useful in cases of B. Anthracis meningitis, can be used totreat the lethal factor toxin component of pulmonary anthrax.Furthermore, the claimed invention is intended to apply to otherpathological conditions which involved the LF both presently known andunknown.

EXAMPLE 3 Antifungal Activity of 4-Butyl-2-Thophenecarboxylic AcidHydrazide and Analogues

[0427] The chelating agents of the present invention can also be used tocontrol topical and systemic fungal infections in various diseaseconditions. It has been documented that copper, zinc, and iron areessential metal ions in critical fungal enzymes such as superoxidedismutase, metalloproteases, and ribonucleotide reductase, respectively.In general, the increased proliferation rate of fungus is the result ofavailability of nutrients and transition metal ions which activateessential metalloenzymes. The administration of the specific chelatingagents of the instant invention can prevent unwanted fungalproliferation by blocking the activity of these metalloenzymes. Thechelating agent can be administered orally or parenterally in dosesdescribed elsewhere in this application.

[0428] Antifungal agents are naturally occurring compounds or syntheticthat have in vitro and in vivo activity against yeast, mold or both.Since fungi and mammalian cells are eukaryotic, and the antifungalagents inhibit synthesis of proteins, RNA and DNA, they have toxiceffects in the mammalian host.

[0429] The high incidence of toxicity among antifingal agents results inthe fact that there are only a few antifungal agents currently used inhuman treatment. However, the number of fungal diseases has increased inthe past 30 years, especially among immunocompromised patients which areat high risk for life-threatening mycosis. Topical use is also increaseddue to the fungal infections of the nails.

[0430] The agents of this invention can be used use as novel antifungalagents in animal and human mycoses, in mycotic infections, and inemerging fungal infections in immunocompromized patients. They can beused in superficial and systemic mycosis. For example, they can be usefor topical use in nail infections and for systemic use inimmunocompromized patients such as those having AIDS or lung transplantfungal infections.

[0431] If some of the selected agents show untoward toxicities inclinical trials, such as lysis of erythrocytes, they can be modified byincorporating the agents of this invention in lipid bilayers, in orderto protect the erythrocytes and thus make the drug available as asystemic agent. The agents of this invention and in particular thelipophilic 4-butyl/pentyl-substituted derivatives of THA-hydrazide canbe incorporated in multilamellar liposomes which can contain specificratios of lipids such as phospholypids dymyristoyl phosphatydylcholine(DMPC) and dimyristoyl phosphatydylglycerol (DMPG) in a 7:3 ratio. Thislipid-modified antifungal agents can be administered in doses equivalentto those described elsewhere.

[0432] It will be appreciated by those skill in the art that theinventors have disclosed the best mode of the invention for treatment offungal diseases. Therefore, the foregoing specifications andaccompanying drawings are intended to be illustrative of this exampleonly.

EXAMPLE 4 Antiparasitic Activity OF 4-Butyl-2-Thiophenecarboxylic AcidHydrazide and Analogues

[0433] Practical, effective, and inexpensive drugs are needed to treatparasitic infections, particularly malaria. The practical, effective,and inexpensive chelating agents of the present invention can be used tocontrol parasitic infections in various disease conditions in animalsand man.

[0434] It has been documented that copper, zinc, and iron are essentialmetal ions in critical parasitic-encoded enzymes such as zinc fingerribosomal proteins (MPS/S27), Zn-dependent metalloproteases, andribonucleotide reductase, respectively. The administration of thespecific chelating agents of the instant invention can prevent unwantedparasitic proliferation by blocking the activity of thesemetalloenzymes. The chelating agent can be administered orally orparenterally in doses described elsewhere in this application.Furthermore, the drugs developed for other clinical indications in thisinvention can be used to treat animals and men hosting parasites. Thesedrugs can be effective for multi-drug resistant parasites. These newdrugs can also be use for prophylaxis.

[0435] In addition to malaria, other protozoans within the scope of thesubject invention are intracellular parasites of man and animals, forexample, Plasmodia, Toxoplasma, Amoeba histolytica, and Trypanosomas. Ofcourse, the agents of this invention can be used to treat helminticdiseases such as filariasis.

[0436] Parasitic infections caused by pathogenic protozoa affect a largeproportion of people of this planet and results in a substantial healthand economic burden. Military operations, world travel andless-developed countries promote the infections by these agents. Malariaaffects more than 500 million people and causes about 2 million deatheach year. This disease affects children, pregnant women andimmunocompromised individuals such as those having AIDS.

[0437] Malaria is an enormously negated disease. Malaria, particularlythe clinical form produced by Plasmodium falciparum, is the mostdevastating disease, and thus it causes high morbidity and mortality.Chemotherapy is the most cost-effective way to control most parasiticinfections, including malaria. Many of the drugs used to combat suchinfection have been in use for over 50 years. Therefore, one of themajor problems is resistance to the agents used for chemotherapy.

[0438] New or superior pharmaceuticals are urgently required to controlsystemic infections such as Malaria, Chagas' disease, visceralLehismaniasis, etc. These new drugs are needed to prevent thedevelopment of drug resistance. Because protozoa proliferate rapidly inthe host, they develop resistance to drugs readily.

[0439] The complex life cycle of malaria makes it difficult to attackthis parasite. At each stage the parasite produces different proteins.Thus, to kill malaria, a drug must target different proteins atdifferent parasitic life stages. However, from gene data banks we haveidentified a class of metalloprotein genes essential for parasitesurvival that are expressed at all stages of the life cycle of malaria.These genes are involved in protein synthesis and fat production and areessential for parasite survival. Furthermore, these genes aresufficiently different form human genes, are zinc finger proteins andparasite transition metal ion-dependent metalloproteinases that can betargets for the new drugs of this invention

[0440] The development of economic, safe and effective broad-spectrumagents to treat parasitic diseases is one of the purposes of thisinvention. The method is based on the actions of the compounds presentedhere on essential parasitic-encoded zinc finger proteins such asribosomal protein MPS/S27 and/or parasitic metalloproteinases. Purifiedor synthetic parasitic metalloprotein targets for drug action can bedeveloped for rapid, automated in vitro procedures to select the bestpossible drug molecule presented in this invention. The agents of thisinvention have broad-spectrum activity against all developmental stagesof the parasite but particularly the proliferating stages. The agent canbe used for mass chemotherapy orally and it will not induce drugresistance.

[0441] This example deals with the properties and uses of4-butyl-THA-hydrazide and derivatives thereof to treat and preventmalaria caused by four species of Plasmodium, of which P. falciparum isthe most fastidious. This agent acts in the asexual erythrocytic stagesof malarial parasites and in the latent tissue forms of this parasite.Since no single agent has successfully controlled the emergence ofdrug-resistant strains, this drugs will be used in multidrug regimens.The agents of this invention act on the primary tissue forms ofplasmodia and can be used in prophylaxis. Due to the low toxicity, thisagents can be use to produce a suppressive cure, eliminating allparasites from the host.

[0442] There are diseases such as Trypanosomiasis and Leishmaniasis thataffect millions of people in the tropics. Effective antiprotozoal drugsfor treatment of major protozoal infections such as AfricanTrypanosomiasis (sleeping sickness), Chagas' disease, and visceralLeismaniasis are still lacking. Many of the drugs used are toxic andproduce resistance.

[0443] The chelating agents of the instant invention can be as effectiveor superior to benznidazole, the compound currently in clinical use forthe suppression of the reproduction of epimastigotes of Trypanosomacruzi, the protozoa that causes Chagas' disease. The mechanism of actionof the anti-parasitic chelating agents is in intracellular sites of theepimastigote involving iron, copper, or zinc neutralization. The targetprotein can be a small evolutionary conserved parasite-encoded zincfinger protein such as the MPS/S27 ribosomal protein from T. cruzi orother essential parasitic metalloenzymes. The results indicate thatcertain degree of hydrophobicity is necessary for the agents of thisinvention to penetrate the parasites (FIG. 7). The drugs of thisinvention, particularly the hydrophobic derivatives can also be used totreat toxoplasmosis and cryptosporidiosis which are common in AIDSpatients.

[0444] The development of the antiprotozoal drugs of this inventionwhich are specially designed chelating agents to selectively disruptzinc finger proteins and inactivate zinc-metalloproteases critical forthe metabolism of the parasite should provide a new generation of drugsthat can be used in the treatment of the parasitic diseases delineatedabove.

[0445] It will be appreciated by those skill in the art that theinventors have disclosed the best mode of the invention for treatment ofparasitic diseases. Therefore, the foregoing specifications andaccompanying drawings are intended to be illustrative of this exampleonly.

Neurodegenerative Diseases Inhibition of Polymerization of Amyloid,Prions and Other Transition Metal Ion-Dependent Monomeric PrecursorProteins by 4-Butyl-2-Thiophenecarboxylic Acid and Analogues

[0446] The chelating agents of the present invention can also be used tocontrol metal-dependent protein aggregation in various diseaseconditions such as Alzheimer's, Prion diseases and other diseasesinvolving protein aggregation. It has been documented that Fe2+ and Cu2+are involved in abnormal protein aggregation in neurons. The increasedformation of abnormal aggregates in neurons results in apoptosis. Theadministration of the specific chelating agents of the instant inventioncan prevent unwanted protein aggregation in neurons. The chelating agentpenetrates the blood-brain barrier and can be administered orally orparenterally in doses described elsewhere in this application.

[0447] The most common neurodegenerative (ND) diseases are Alzheimer'sdisease (AD), Huntington's disease, and Prion diseases. Although at theclinical and neuropathological level these diseases are distinct, at thetransition metal ion (TMI) level they may have certain unifyingfeatures.

[0448] AD is a progressive and largely untreatable disease. The benefitsthat drugs produce are marginal if any. Most patients after briefinitial gains, merely decline more slowly. Thus, the treatmentspresently available for AD are symptomatic and do not alter theprogression of the disease.

[0449] Protein aggregation in neurons is a key feature of severalincurable neurodegenerative diseases of adulhood such as Alzheimer's,Huntinton's, and spongiform prion-induced encephalopathy. Elucidatingthe mechanisms of protein aggregation and development of pharmacologicalanti-aggregation agents is important to the development of therapies forthese diseases.

[0450] The development of toxic protein aggregates in these diseases isa nucleation-dependent process that can be inhibited by drugs includingthose of this invention. Insoluble, protease resistant, fibrillarprotein aggregates have previously been found in the brains of patientswith AD and also in certain transgenic mice animal model systems. Thesediseases develop slowly, as would be expected if aggregation were theprimary cause.

[0451] A therapeutic strategy is the development of specific chelatingagents that inhibit TMI-dependent aggregation of specific brainproteins. 4-butyl-2-THA-hydrazide and pharmacologically acceptablederivatives thereof in the dosages delineated above can be used toprevent the formation of aberrant protein aggregates that are induced bythe presence of Fe2+, Cu2+, and other TMI, including toxic Al³⁺ that hasbeen involved in the pathogenesis of AD. These agents can be used forthe treatment of AD and other ND diseases involving TMI-dependentprotein aggregates.

[0452] The following examples refer to the use of the agents of thisinvention to inhibit TMI-dependent amyloid aggregation in the brain ofpatients with AD and Cu2+-dependent Prion aggregation in spongiformencephalopathy. Furthermore, it is also conceivable that some of theagents of this invention may be used to prevent the progression ofParkinson's disease that is associated with oxidative damage to neuronsinduced by Fe2+ and Cu2+ accumulation in specific areas of theencephalon.

EXAMPLE 1 Design and Testing of 2-Thiophenecarboxylic Acid AnaloguesInhibitors of Fibril Polymerization in Neurons

[0453] Amyloid beta-peptide (Ab) is a 40 amino acid proteolytic fragmentof amyloid precursor protein (APP). Neuropathologic and transgenicmodeling experiments implicate the increased expression and accumulationof Ab as a necessary step in the pathogenesis of AD. Under physiologicalconditions, monomeric Ab is a nonpathogenic molecule generated duringmetabolism of APP. Under pathological conditions, Ab undergoes achemical process of polymerization that produces amyloid fibrils thatare extremely toxic to neuronal cells. The mechanism of amyloid fibriltoxicity is associated with perturbations of transition metal ionmetabolism (Cu2+, Zn2+, Fe2+), Ca2+ homeostasis, and oxidative damagethat leads to neuronal apoptosis. Although the mechanism ofpolymerization is not clearly understood, suppression or prevention ofthe transition of Ab from monomeric to highly toxic polymeric forms hasbeen identified as a target in the development of therapies for AD. Thisexample details our design and testing of chelating agents inhibitors ofpolymerization of Ab as potential therapeutics for AD.

[0454] One solid hypothesis about how to restrict A-beta's accumulationis the use of chelating agents. This hypothesis was based on theobservations that zinc, copper, and iron can rapidly induce theconversion of A-beta into amyloid. High concentrations of these threetransition metals in Alzheimer's plaques have been reported by numerousinvestigators. It was demonstrated that copper and iron were extremelyactive in that they accelerated both A-beta polymerization andA-beta-related free radical and oxidative stress to neurons.

[0455] Thus, it appears that would be beneficial to have a specificchelating agent that removes metals from the specific brainmicroenvironment and could be incorporated in a pharmaceutical form thatwill penetrate the brain. It is conceivable that by removing the TMI,the A-beta would not cluster in the neurons and that the amyloid plaqueswill be dissolved and/or the aggregates will not form.

[0456] Several groups have published data on approaches to developchelating agents inhibitors of amyloid fibril formation. A number ofchelators such as pyridine carboxylates work quite well in vitro asdemonstrated by the fact that they prevent A-beta's clumping in a testtube, and some chelators even dissolved amyloid taken from autopsiedAlzheimer's brains. However, these chelators showed sharp dose-responsecurves consistent with the fact that they are too harsh of a treatmentto have the potential for chronic therapeutic applications in animals orhumans. The fact is that these carboxylic acid and derivatives removedtoo much metal from the body, depleting vital amounts of TMI and thusdecreasing the cells' energy with resulting apoptosis. These carboxylicacid chelators and derivatives are strong chelators that when added tofoods, it ensures that metal-dependent types of bacteria will notsurvive. We turn, instead, to more specific and less active TMI-bindingagents, with high lipid membrane permeability. These compound which arethe subject of this invention can be developed into new drugs based onour compound's potential as a metal-ousting anti-amyloid therapy.

[0457] We have begun to work with novel chelating agents derivatives of2-TH acid that show sigmoidal dose-response curves, have wide effectivedose-ranges (1 to 20 uM), and high penetrability in brain slices due totheir lipophilic side chains. This example describes the essentialelements of the design and testing of such gentle compounds that can beuse to prevent polymerization of Ab intracellularly in neurons withoutinducing toxicity or apoptosis.

[0458] Amyloid formation can be monitored by several techniques,including electron microscopy, light scattering, cellular toxicity,nucleation and extension assays. In the extension assay, the primaryeffect of an inhibitor is to slow the rate of fibrillogenesis. Thechelating agents of this invention can prevent the polymerization of Abin extension assays at uM concentrations.

[0459] 2-TH acid and derivatives are drugs that act by attacking thebasic process of AD. They block the formation of amyloid fibrils. Thus,2-TH acid and pharmacologically acceptable derivatives thereof in thedosages delineated above can be used to prevent the formation ofaberrant TMI-dependent protein aggregates in AD. This exampledemonstrates the possibility of developing therapeutic agents that mayalter the course of AD by preventing neuronal death These drugs shouldhelp in maintaining or improving cognition, memory and global function

[0460] The development of the anti-amyloid drugs of this invention whichare specially designed chelating agents to selectively disrupt Abaggregation should provide a new generation of drugs that can be used inthe treatment of the AD and possibly other diseases discussed below.

[0461] It will be appreciated by those skill in the art that theinventors have disclosed the best mode of the invention for treatment ofprotein aggregation diseases. Therefore, the foregoing specificationsand accompanying drawings are intended to be illustrative of thisexample only.

EXAMPLE 2 Prions Bind Copper and are Susceptible to Modulation by 2-THAcid and Derivatives

[0462] Computational molecular models, optical spectroscopy and nuclearmagnetic resonance (NMR) investigations of synthetic prions (PrP)peptides have demonstrated that these proteins are able to bind copperin a specific fashion. The highly flexible NH2-terminus of recombinantPrP is more structured in the presence of Cu2+ ions. Each PrP moleculewas found to bind two Cu2+ ions at pH 6.5. At pH 7.4, four Cu2+ ions arebound to the PrP. Other divalent cations including Co2+, Mg2+, Mn2+,Ni2+, and Zn2+ did not bind to PrP. When Cu2+ binds to PrP a sequencecorresponding to an octapeptide changes configuration and is transformedin an alpha-helix.

[0463] More recently, PrP-deficient mice were found to have lower levelsof Zn/Cu superoxide dismutase (SOD) activity. SOD activity has beenshown to mirror the state of copper metabolism. Further resultsindicated that PrP might function as a Cu2+ binding protein. Both Cu2+and Zn2+ ions have been reported to modify the structure of theN-terminal PrP. However, the molecular basis for these changes are notknown.

[0464] Disturbances in Cu2+ homeostasis leading to dysfunctions of thecentral nervous system are well documented both in animals and man. Forexample, Wilson's disease is a genetic disease that involves abnormalcopper metabolism. More recently, cuprizone, a Cu2+ chelating agent, hasbeen used in mice to induce neurophathological changes similar to thoseobserved in Prion diseases.

[0465] If Cu2+ is needed to maintain normal Prion configuration,organometalic derivatives of 2-TH acid such as Cu2+-2-TH acid may beused to neutralize aggregation of prions induced by Cu2+ depletion. Forexample, Cu2+-2-TH acid and derivatives may be used to control thealpha-helix configuration of PrP and thus, they may prevent thedeleterious effects of these abnormal proteins.

EXAMPLE 3 Brain Iron in the Pathogenesis of Parkinson's Disease and itsNeutralization by the TMI Chelators of this Invention

[0466] A central role of iron in the pathogenesis of Parkinson's disease(PD) is related to its increase in substancia nigra pars compactadopaminergic neurons and reactive microglia. The deleterious effects ofiron in neurons is related to its capacity to enhance production oftoxic reactive oxygen radicals in these cells. The critical role ofFe2+/3+in the pathogenesis of nigrostriatal injury is also reinforced bythe ability of Fe2+/3+ to induce aggregation of alpha-synuclein andtoxicity in these cells. Because many neurodegenerative diseases showincreased accumulation of Fe2+/3+at the site of neurodegeneration, it isbelieved that maintenance of cellular iron homeostasis is critical forthe survival of neurons. Furthermore, both oxydative stress andaccumulation of iron are involved in the induction of apoptosis in PD.Prevention of the production of free radicals may be of therapeuticimportance in PD. The neutralization of Fe2+/3+in the neurons can beaccomplished by the specific TMI chelators of this invention such as4-butyl-2THA-hydrazide which penetrates the blood brain barrier.

Inflammatory Response

[0467] Inflammation is an essential pathophysiological response to alarge number of diseases which can affect all tissues and organ systems.Diseases involving inflammation can be acute and fatal whereas othersare chronic. The development and maintenance of inflammation iscontrolled by a complex network of humoral and cellular factors.Paracrine hormones such as the eicosanoids are derived from theoxidative metabolism of arachidonic acid which produces importanthormones that include prostaglandins (PG), thromboxanes (TX),leukotrienes (LT), and lipoxins (LX).

[0468] The inflammatory response has evolved as a humoral and cellulardefense system to protect the tissues when exposed to injury such aschemical, viral, etc. For a number of reasons, such response may becomeundesirable. The administration of the specific chelating agents of theinstant invention can prevent unwanted inflammation. The chelating agentcan be administered orally or parenterally in doses described elsewherein this application.

[0469] Furthermore, the claimed invention is intended to apply to otherpathological conditions which involved inflammatory responses bothpresently known and unknown. It will be appreciated by those skilled inthe art that the inventors have disclosed the best mode by which theypresently understand 2-TH acid and its derivatives, to function incontrolling inflammatory responses. However, the scope of the appendedclaims is intended to include other mechanisms of action, both presentlyknown and unknown, which include metal ion containing proteins asmediators in inflammatory responses including, but not limited to,parasitic diseases such as toxoplasmosis, malaria, and lehismaniasis. Itis worth noting here that arteriosclerosis has an inflammatory andproliferative component which may be blocked by the methods andcompositions of the instant invention.

[0470] It is also important to recognize that 2-TH acid inhibitors mayoffer several other potential health benefits, including analgesia, andpossibly preventive effects against cognitive disorders (e.g. Alzheimersdisease), colorectal cancer, and arteriosclerosis formation. Theusefulness of this invention has the potential for significant publichealth rewards.

[0471] It will be appreciated by those skill in the art that theinventors have disclosed the best mode by which they presentlyunderstand 2-TH acid and its derivatives, to function in the control ofpathological inflammatory responses.

[0472] There are numerous diseases involving inflammatory responses thatmay be susceptible to the compounds and methods presented in thisinvention. The following examples illustrate the use of the agents ofthe instant invention in common human inflammatory diseases.

EXAMPLE 1 The Compounds and Methods of the Present Invention can be Usedto Inhibit the Action of the DnaJ Zinc Finger Proteins When They areExpressed in Inflammation at Pathologically High Levels

[0473] Prokaryotes and eukaryotes express numerous heat shock proteins(Hsps) in response to stress, including heat shock, exposure to heavymetals, hormones and viral infections. Hsps also mediate physiologicaland pathological inflammatory responses. Hsps are involved in cancer,such as colon and breast cancers.

[0474] The stress response that includes numerous forms of physiologicaland pathological stress is involved in viral infection. A prominentfeature of this response is the synthesis of a discrete set of zincfinger proteins, known as the heat shock proteins, which at present aredenoted molecular chaperons. During infection by certain viruses, heatshock proteins act as intracellular detectors that recognize malfoldedproteins. Researchers have found that certain DNA viruses are able toactivate heat shock proteins. For example, the Hsp70 (DnaK) is inducedby adenovirus, herpes virus, cytomegalovirus, and other viruses.

[0475] One of the most interesting proteins involved in the viralinfection response is the DnaJ, a heat shock protein which functions inthe control of protein folding within the cell (FIG. 12). DnaJ proteinscontain two CCCC zinc fingers, defined by the J domain, which isessential for stimulation of the Hsp70 ATPase activity (FIG. 12). Thus,the results suggest that there may be a relationship between the stressresponse and the cytopathic effects of certain viruses such as herpesviruses.

[0476] The response of cells to stress, such as exposure to UVradiation, chemicals, or viruses is also associated with the inductionof heat shock proteins. Hsp70 has a protective role in inflammation,infection, and regulatory roles in cytokine biosynthesis. Hsp70 existsin the cells in equilibrium between its free state, in the cytoplasm,and its bound state, protecting proteins in the nucleolus, interactingwith ribosomal proteins to either refold some of the unfolded ribosomalproteins or by solubilizing the denatured ribosomal proteins tofacilitate their use and increase the turnover rate. During release as aresult of the heat shock, and as the nucleolus begins to recover itsnormal activities, a significant proportion of Hsp70 returns to thecytoplasm. This protein-protein interaction may have profoundimplications for viral replication.

[0477] Thus, cellular inflammatory responses to viral infection are partof the organism defense against viruses. Zinc finger proteins,therefore, may be a key to the control of the cellular inflammatoryresponse. Agents which can modify the zinc finger heat shock proteinsmay be useful in controlling the stress response.

[0478] The compounds and methods of the present invention can be used toinhibit the action of the DnaJ zinc finger proteins when they areexpressed in inflammation at pathologically high levels. By blocking theDnaJ zinc finger proteins, the resulting conformational change shouldinhibit the ATPase activity. This inhibition of the DnaJ zinc fingerdomain which is required for enzyme activity (FIG. 12) will reduce theinflammatory reaction in cells expressing high levels of this protein.

EXAMPLE 2 The Compounds of the Present Invention can be Used for theSimultaneous Control of Bacterial, Parasite and/or Fungal Cell Growthand the Inflammatory Responses Induced by These Biological Agents

[0479] It is believed that the inflammatory response to bacteria,parasites or fungi is associated with the induction of heat shockproteins. Hence, the compounds and methods of the present invention canbe used to block inappropriate and excessive cellular inflammatoryresponse caused by induction of HSP by for example by P. acnes, thebacteria that produces acne. The chelating agents of this invention cansimultaneously block the zinc finger proteins enzymes of theinflammatory response induced by the bacteria of common acne and inhibitthe growth of this bacteria. Similarly, the agents of this invention cancontrol bacterial, parasite and/or fungal growth and the associatedinflammatory responses in other disease conditions.

EXAMPLE 3 Alzheimer's Disease and Inflammation

[0480] A chronic inflammatory reaction with activated microglia cellsand astrocytes is a constant feature of AD. The inflammatory componentis triggered by neuronal apoptosis in combination with the production ofcytokines by microglia. Experimentally, the cyclooxygenase inhibitoribuprofen decreases cytokine-induced amyloid beta production in neuronalcells. The data suggests that the simultaneous reduction of Abetaproduction and the associated inflammatory response by anti-inflammatoryagents may be useful to prevent and treat AD. Thus, pharmacologicallyacceptable doses of the 2-TH acid and derivatives with the capacity tocross the blood-brain barrier can be use to treat both the Abetaaggregation and the inflammatory component of AD.

EXAMPLE 4 Nonsteroidal Anti-Inflammatory Drugs and Colorectal CancerChemoprevention

[0481] It has been shown that there is a 40% to 50% reduction inmortality from colorectal cancer in persons using nonsteroidalanti-inflammatory drugs (NSAIDs) on a permanent basis. NSAIDs, such asaspirin and ibuprofen, inhibit both cyclooxygenase-1 (COX-1) andcyclooxygenase-2 (COX-2) activity. COX-2 levels are increased in anumber of solid tumors. Thus, COX-2 may be a molecular target for cancerprevention and/or treatment. The anti-inflammatory properties of NSAIDsare most likely due to their inhibition of cyclooxygenase enzymes. Theseenzymes catalyze key steps in the conversion of arachidonic acid toprostaglandins and other eicosanoids. Long term NSAIDs use results in anincrease gastrointestinal bleeding, even at low doses of the drug. Thisside effect increases in the elderly patients that are at higher riskfor colorectal cancer.

[0482] It is well established that the inflammatory cells such asfibroblasts and lymphocytes, release growth factors such as FGF, TGFalpha, interleukins, etc. These factors stimulate the growth ofsurrounding cancer cells. Thus, it is the contention of the inventorsthat inhibition of inflammatory responses, which include inactivation ofmetalloproteins and arrest of proliferation of cells such asfibroblasts, by using the agents presented here should be useful in theprevention and treatment of cancer.

[0483] By inhibiting the inflammatory response, the growth factors andcytokines generated by the inflammatory cells of the stroma, will not beavailable for stimulation of tumor growth. Thus, the agents of thisinvention work in at least three levels: 1) Inhibition of growth offibroblasts and other inflammatory cells; 2) inhibition of angiogenesisand 3) inhibition of cancer cell growth.

[0484] Depending on the dose and duration of drug required, the sideeffects of the chemoprenventive agents of this invention will be low toachieve the desire result, because the absolute risk of colorectalcancer in the general population is low. Combination of agents forchemoprevention of cancer may provide a much more effective approach forlong term cancer prevention.

EXAMPLE 5 Transplant Rejection and Inflammatory Responses

[0485] The chelating agents of the present invention can also be used tocontrol transplant rejection in various disease conditions. It has beendocumented that matrix metalloproteases (MMPs) are actively involved intransplant rejection (FIG. 13). Furthermore, transplant rejection has astrong inflammatory component. The administration of the specificchelating agents of the instant invention can prevent both unwantedexpression of MMPs and inflammation. The chelating agent can beadministered orally or parenterally in doses described elsewhere in thisapplication.

[0486] Organ transplant is a well-established therapy for many forms ofirreversible failure of many organs. However, the success of solid-organtransplantation is a function of the continuous administration of toxicand non-specific immunosuppressive agents, which carry the risks ofopportunistic infections, malignancy and many drug-specific side effectssuch as hypercholesterolemia and hyperglycemia. In most instances, acuterejection can be overcome by specific treatments. However, the adverseeffects of chronic immunosuppression, graft degradation and loss causedby chronic rejection continue to be a problem.

[0487] During organ rejection, immune responses result in persistentperivascular inflammation (FIG. 13). Cytokines, chemokines, and growthfactors released by the inflammatory and repair processes stimulate theproliferation of smooth muscle cells (SMC) (FIG. 13). This results inthe migration of myocytes from the media to the intima, resulting in theformation of arteriosclerotic lesions (FIG. 13). Ultimately, vascularischemia and the subsequent development of interstitial fibrosischaracterize chronic allograft rejection.

[0488] SMC proliferation activate the production and release of SMCmatrix metalloproteinases in the vascular wall resulting in thedigestion of surrounding extracellular vascular matrix which allows SMCcells to migrate from the media to the intima (FIG. 13).

[0489] Several therapeutic approaches have been used to control theintimal hyperplasia that occur in chronic transplant rejection. Onestrategy consists in the inhibition of smooth muscle cell proliferationwhich results in decreased ischemia, infection, and perivascularinflammation. Another strategy is to prevent smooth muscle cellmigration.

[0490] Several studies have shown that smooth muscle migration can beprevented by the expression of a gene encoding an inhibitor of MMPs. Theresults showed that this gene therapy significantly prevented smoothmuscle cell migration and neointimal formation.

[0491] The agents of this invention can be used to stop chronic organrejection by inhibiting at least two pathological processes: 1) theproliferation of smooth muscle cells (SMC) and 2) The activation ofmatrix metalloproteinases in the vascular wall which allows smoothmuscle cells to digest the surrounding extracellular matrix and migratefrom the media to the intima (FIG. 13).

[0492] The agents of this invention could be useful for prevention ofchronic rejection because these agents can arrest SMC proliferation anddevelopment of smooth muscle cell migration, neointimal formation, andsubsequent interstitial fibrosis and inflammation which characterizechronic allograft rejection.

EXAMPLE 6 Zinc Induced Neuronal and Glial Cell Death and Brain Injury

[0493] In addition to the essential role of zinc as a structural orcatalytic element of many proteins, in the central nervous system theabundant zinc has an additional specialized role as an intracellularsignaling messenger. In this capacity, zinc is released by neuralactivity at numerous central excitatory synapses. Despite its generallack of toxicity, the data indicates that zinc can became a pathogenicmetal that mediates neuronal death in certain neurological diseasestates. More specifically, following transient global ischemia, Zn2+translocates form nerve terminals into the cell bodies of alteredneurons. This translocation precedes neuronal degeneration by apoptosis,and the interruption of this transit by intracerebroventricular (icv)injection of the chelator EDTA bound to Ca2+ reduces neuronal apoptosis.Zn2+ released in excessive amounts, as is the case of glutamate,contributes to the development of cerebral infarctions followingischemia, seizures, or head trauma Reduction of extracellular zincaccumulation and/or intracellular vulnerability to toxic zinc exposureprovides a novel therapeutic approach to prevent pathological neuronaldeath. A major problem of the currently available zinc chelators isachieving adequate access to the CNS. Some of the agents of thisinvention are hydrophobic and thus are able to permeate the blood-brainbarrier. Thus, pharmacologically acceptable doses of the 2-TH acid andderivatives with the capacity to cross the blood-brain barrier can beuse to control Zn2+ released in excessive amounts in cerebralinfarctions following ischemia, seizures, or head trauma.

EXAMPLE 7 Inflammation and Inhibition of Leukotriene A₄ Hydrolase by2-TH Acid and Analogues High Specificity is Achieved by Substituting thePosition 3 of 2-TH Acid With a Hydrophobic Chain that Interacts with theHydrophobic Pocket of the Target Enzyme

[0494] The chelating agents of the present invention can also be used toinhibit specific enzymes involved in inflammation such as Leukotriene A₄hydrolase (LA₄H)in various disease conditions (FIG. 14). This enzyme isa zinc-dependent metalloproteinase. LTA₄ hydrolase belongs to the M1family of metallopeptidases. It is of practical medical interest toinhibit an enzyme that is involved in inflammatory diseases. Theadministration of the specific chelating agents of the instant inventioncan prevent unwanted inflammatory responses mediated by this enzyme. Thechelating agent can be administered orally or parenterally in dosesdescribed elsewhere in this application.

[0495] Based on the zinc signature, sequence homology and aminopeptidaseactivity, LTA₄ hydrolase has been classified as a member of the M1family of zinc-metallopeptidases. Leukotriene (LT) A₄ hydrolasecatalyzes the committed step in the biosynthesis of LTB4, a classicalchemoattractant and immune-modulating lipid mediator involved ininflammation, host-defense against infections, and systemic,PAF-mediated lethal shock. LTA₄ hydrolase is a bifunctional zincmetalloenzyme with a chloride-stimulated arginyl aminopeptidaseactivity. When exposed to its lipid substrate LTA₄, the enzyme isinactivated and covalently modified in a process termed suicideinactivation, which puts a restrain on the enzyme's ability to form thebiologically active LTB4.

[0496] In this section of this application we show that the activeenzyme center can be specifically inhibited by the agents of thisinvention (FIG. 14). The proposed mechanism of inhibition by the agentsof the invention is presented. The zinc site and catalytic residues areinhibited by 2-TH acid analogues (FIG. 14).

[0497] The leukotrienes (LT) are a group of lipid compounds with potentbiological activities. They are involved in inflammatory responses andallergic disorders. These compounds are derived from the metabolism ofarachidonic acid. Leukotrienes were originally isolated from leukocytes.The bone marrow cells are the main producers of LT, particularly PMNleukocytes, monocytes and tissue macrophages and mast cells. LT are verypotent chemoattractans for neutrophils and recruit inflammatory cells tothe site of injury. They increase leukocyte adhesion to endothelialcells of blood vessels and have potent vasoconstriction activity insmooth muscle. They also induced bronchoconstriction. LT are chemicalmediators of inflammatory and allergic reactions in diseases such asrheumatoid arthritis and bronchial asthma.

[0498] LTA₄ is widely distributed in almost all mammalian cells, tissuesand organs examined. In the blood, neutrophils, monocytes, lymphocytesand erythrocytes are rich sources of the enzyme. LTA₄ is inactivated andcovalently modified by its substrate. The competitive inhibitor bestatinprevents the covalent binding of LTA₄ to the enzyme, showing that itoccurs at the active site.

[0499] LTA₄ hydrolase contains a zinc site that binds a single catalyticzinc. The similarity to other zinc-metalloenzymes is higher over a shortsegment of the homologous proteins, which contains a consensus sequencefor a catalytic zinc site (H-E-(X)₁₋₃—(X)₁₈₋₁₂₀-E). This clearly showsthat LTA₄ hydrolase is a zinc containing enzyme and that His-295,His-299, and Glu-318 are the zinc binding ligands. Of great interest forthis invention is the fact that the enzyme can be inactivated by thezinc chelator 1,10-phenanthroline which converts the enzyme in anapoenzyme (minus Zn). Addition of stoichiometric amounts of zincrestores the enzymatic activity. Thus, the identification of LTA₄hydrolase as a member of a family of zinc metalloproteinases makes thisenzyme a target for the agents of this invention.

[0500] Based on the zinc signature, sequence homology and aminopeptidaseactivity, LTA₄ hydrolase has been classified as a member of the M1family of metallopeptidases. Thus, LTA₄ hydrolase is related to numerousother zinc proteases that are present in many organism from bacteria tomammals.

[0501] It was also demonstrated that LTA₄ hydrolase possesses alipid-binding pocket, which can be occupied by LTA₄ or by the drugs ofthis invention (FIG. 14). The fact that LTA₄ hydrolase belongs to afamily of zinc proteases opened up novel possibilities of using theagents of this invention as specific enzyme inhibitors. Captopril, azinc chelator inhibitor of the angiotensin converting enzyme alsoinhibits LTA₄ hydrolase. Captopril inhibits the enzyme at the low uMrange. Captopril is extensively used as an antihypertensive agent inhumans and is metabolically stable after oral administration.

[0502] Based on molecular modeling, the reaction mechanisms, andinhibitor-enzyme interactions for zinc hydrolases, we have developedselective inhibitors for this enzyme (FIG. 14). For example, the4-pentyl-2-THA-thiosemicarbazone that has a hydrophobic tail at position4, was found by molecular modeling to be an effective inhibitor of LTA₄hydrolase in the low uM range. This lipophilic compound can also be apotent and selective inhibitor of LTA₄ hydrolase in leukocytes. Thecompounds of this invention were designed to inhibit LTA₄ hydrolase andshould be orally active.

[0503] The human LTA₄ hydrolase has been modeled by the inventors. Thethree dimensional structure revealed a protein with 3 domains whichtogether form a deep cleft harboring the zinc catalytic site. Thepurpose of the following description is to cover some of the mostsalient molecular details of the LTA₄ hydrolase that are pertinent tothis invention. It is not within the scope of this invention to describethe intricate relationship between the substrate and the product.

[0504] We have generated a model to identify structural and functionalelements of the active site and surroundings. This in turn generatedinformation that was used in the design of potent and specific enzymeinhibitors of this invention (FIG. 14). These compounds can induce smalland large changes in the structure leading to a conformationalalteration that affects the tertiary structure of the enzyme leading toits degradation by other proteases.

[0505] As can be investigated in the data banks, the zinc site in LTA₄is located at the bottom of a cleft. The metal is bound to three aminoacid ligands, His-295, His-299, and Glu-318. In FIG. 14 the zinc shownis bound to the 4-pentyl-2-THA-thiosemicarbazone, which creates apentavalent covalent coordination between the drug and the active site.

[0506] The residues lining the pocket are conserved because they belongto the active center. One patch of the cavity is hydrophilic, which canmake direct electrostatic interactions with the positive changes of theinhibitor of this invention, in agreement with the fact that freecarboxylic acid of LTA₄ is required for catalysis. Furthermore, andadditional hydrophobic cavity located in the vicinity of the catalyticzinc is the LTA₄ biding site. The zinc acts as a weak Lewis acid toactivate and open the epoxide ring of LTA₄. In accordance to this fact,the 4-pentyl-2-THA-thiosemicarbazone inhibitor also binds to thehydrophobic cavity by the 5-pentyl side chain (FIG. 14).

[0507] The shape and curvature of the hydrophobic regions at position 4of the agents of this invention indicate the chemical strategy for thecreation of an effective inhibitor of LTA₄ hydrolase. The inhibitorshown in FIG. 14 is the 4-pentyl-2-THA-thiosemicarbazone(4-P-2-THA-TSC).

[0508] The modeled 4-P-2-THA-TSC inhibitor molecule adopts a bent shapethat fits very well with the architecture of the binding pocket. Hencethe critical double bond (hydrophobic-Hydrophylic-metal) geometry fitswell with the architecture of the binding site. Considering the dataobtained from modeling, the inhibitors of this inventions are highlyspecific. Thus, differential mapping using information available in databanks plus the characteristics of the inhibitors of this invention leadsto a general formula to select potential specific inhibitors of this andother zinc metalloenzymes (FIG. 14).

Heavy Metal Poisoning

[0509] The invention relates to the treatment of heavy metal toxicity inanimals and humans. More specifically, the invention relates to the useof metal chelating agents, including furoic acid, 2-TH acid and theirderivatives, analogues and related chemicals falling within thedefinition of the formulas shown in FIGS. 1 and 2 as pharmacologicalagents to prevent and/or treat toxicity caused by heavy metals. Theprevention and treatment of diseases and toxicities caused by metalssuch as uranium, lead, iron, copper, nickel, and tungsten can beaccomplished by the chelating agents of this invention. The chelatingagent can be administered orally or parenterally in doses describedelsewhere in this application.

[0510] The chelating agent is administered to the patient by systemicadministration at the proper dosage by injection, transdermal, rectal,inhalation, intranasally or other pharmacologically acceptable form. Thechelating agent displaces the toxic metal from the protein, binds thetoxic metal, which results in an inactive chelate and subsequently iseliminated from the body.

[0511] The environmental metals most deleterious for human health arelead, mercury, arsenic and cadmium. The daily exposure to lead is amajor pediatric concern. All these metals are carcinogenic.

[0512] Heavy metals exert their toxic effects by binding with one ormore ligands essential for normal physiologic functions. Heavy-metalantagonists, denoted chelating agents, are created specifically tocompete with these ligands for the metals. In this form, they preventand reverse toxic effects and enhance the excretion of the metals.

[0513] The chelating agents of this invention have the followingproperties: high solubility in water, stable, penetrate to the sites ofmetal storage, capacity to form non-toxic complexes with the toxicmetals, ability to retain toxic metals at low pH and able to excrete thechelate. Furthermore, they have low affinity for Ca2+.

[0514] The following examples illustrate the use of the chelating agentsof this invention to prevent or treat heavy metal poisoning.

EXAMPLE 1 Prevention of Formation of Abnormal Iron-Finger Proteins

[0515] By molecular modeling, the inventors have determined that it isfeasible to maintain zinc finger metalloproteins in an undamagedzinc-containing configuration by using a combination of specific agentsof this invention and radical scavengers. This combination counteractsthe formation of both aberrant iron-finger proteins and free radicals.Thus, 2-Th acid and pharmacologically acceptable derivatives thereof, inacceptable doses delineated above, can be used to prevent the formationof aberrant iron-finger proteins involved in carcinogenesis and aging.Free radical scavengers include anti-oxidants such as vitamin E,Flavonoids, etc.

EXAMPLE 2 Treatment of Iron and Copper Toxicity

[0516] Acute effects of iron toxicity include hepatic necrosis, coma anddeath. Deleterious chronic effects of iron toxicity usually result fromiron supplementation particularly in the elderly population.Desferoxamine is the iron chelating agent of choice to treat irontoxicity or iron overload. However, this agent is administeredparenterally and has a number of side effects such as hypotension, rash,and analphylatic shock.

[0517] The novel agents of the present invention can be used to chelateand remove the excess iron. The dosage range for this use is 250 mg to6000 mg per day administered intravenously or orally.

[0518] In addition to its use as a chelating agent for the treatment ofcopper, mercury, iron and lead poisoning, the agents of this inventioncan be used in the treatment of Wilson's disease.

[0519] Wilson's disease is a rare hepatolenticular degeneration due toand excess copper in the blood and tissues. Penicillamine is the agentof choice to treat Wilson's disease. However, penicillamine has a numberof adverse reactions including gastrointestinal bleeding due togastritis, and hematological abnormalities.

[0520] The novel agents of the present invention can be use to chelateand remove the excess copper. The dosage range for this use is 250 mg to6000 mg per day administered intravenously or orally.

EXAMPLE 3 Prevention and Treatment of Lead, Cadmium and MercuryPoisoning

[0521] Lead is a ubiquitous metal in the environment as a result of itsnatural occurrence and its industrial use. The primary sources of leadare leaded paint and lead in the drinking water. The major routes ofabsorption of lead are from the gastrointestinal track and therespiratory system. Once lead is absorbed it essentially accumulates inall organs containing metalloproteins. At present, lead poisoning istreated with combination therapy including dimercaprol, EDTA,penicillamine and succimer.

[0522] One of the mechanisms for toxicity of lead is the inhibition of azinc metalloenzyme, gamma-aminolevulinate dehydratase (ALAD), which isalso inhibited by aluminum. In the case of lead, the inhibition occursthrough the substitution of lead for zinc. Thus, the agents of thisinvention can be use to remove lead from this and other metalloproteins.

[0523] Lead poisoning can be prevented or treated by the use of thenovel chelating agents of the present invention. For prevention, theindividual who will be exposed to an environment containing lead in theform of toxic fumes, water, or lead paints can be prophylacticallytreated with the chelating agents of this invention which will chelateand remove from the body the lead prior to the distribution to thetissues. The individual already intoxicated by lead is treated with thenovel chelating agents to remove the lead from the tissues and eliminatethe chelate-lead by the kidneys, subsequently reversing the progressionof the disease. For either indication the broad dose range of 250 mg toless than 6000 mg a day administered intravenously or orally issuggested. Similarly, the same agents and doses can be used in thetreatment of Cadmium and Mercury exposure.

EXAMPLE 4 Prevention and Treatment of Exposure to Stable or RadioactiveHeavy Metals: Uranium, Tungsten and Nickel

[0524] The use of heavy metals such as depleted uranium (DU) andtungsten alloys in military applications worldwide could result inpoisoning from occupational exposure and environmental pollution.

[0525] The use of uranium depleted weapons can result in soldiers withembedded heavy metal shrapnel or poisoning by aspiration of uraniumcontaining gases created in the battlefield. Furthermore, it has beenshown that soluble or insoluble depleted uranium particles cantransformed normal cells to the malignant phenotype. These dataindicates an increase risk of carcinogenesis in exposed individuals.

[0526] The data demonstrates that DU and tungsten are transforming,genotoxic, and ribotoxic agents in vitro. The in vivo effects ofinternalized DU include enhancement of mutagenicity, oncogene activationand tumor suppressor gene neutralization. Similarly, tungsten alloys andnickel were also shown to be neoplastic transforming agents. Tungstenand nickel have been shown to cause genotoxicity, ribotoxicity, andgenomic instability.

[0527] The inventors have determined by molecular modeling that thedeleterious effects of exposure to heavy metals, including depleteduranium, tungsten and nickel can be antagonized by the administration of2-TH acid or derivatives. The chelated heavy metal complex isinactivated and excreted by the kidneys. The compounds of this inventionhave excellent tissue penetrability, including penetration into thecerebrospinal fluid and brain tissues. The inventors' have concludedthat a pharmacologically appropriate dose of the 2-TH acid or derivativethereof, can be effectively used to treat heavy metal toxicity andprevent carcinogenesis in exposed individuals. When prophilacticallyused the novel agents should be able to prevent heavy metal damage tothe cells.

[0528] The novel agents of the present invention can be use to chelateand remove the excess soluble UD, tungsten and nickel. The dosage rangefor this use is 250 mg to 6000 mg per day administered intravenously ororally.

[0529] A kit containing 500 mg capsules of 2-TH acid or derivativesthereof can be carried out by soldiers or other personnel in the fieldwhen contamination by stable or radioactive heavy metals is a risk. Thesoldiers can begin ingestion of the appropriate dose of the chelatorupon exposure to the radioactive heavy metals such as depleted uranium.

Illustrative Preparations Containing Metal Chelating2-Thiophenecarboxylic Acid and Derivatives for the Treatment andPrevention of Specific Disease States EXAMPLE 1 Topical or IntravaginalPreparation of 2-TH Acid in an Absorption Base

[0530] A topical or intravaginal preparation of furoic acid or 2-TH acidin an absorption base is made by incorporating 0.001% to 99.9%,preferably 1% to 50%, most preferably 5% to 20% 2-TH acid into anabsorption base. One preferred embodiment of the topical preparation ismade by dissolving 10% 2-TH acid in deionized water and thenincorporating the solution into an equal amount of Aquaphor on a wt/wtbasis. Further, the 2-TH acid or derivatives can be incorporated into astick for application to the lips to treat herpes infections. It will beappreciated that 2-TH acid derivatives can be used in place of the 2-THacid in the topical preparation. It will be also appreciated that suchpreparations can be used to treat topical conditions such as virusinfections, fungal infections, susceptible bacterial infections,radiation damage, including ultraviolet, medical or atomic radiation,skin cancers or any other condition mediated by the above describedmechanisms.

EXAMPLE 2 Furoic Acid and 2-TH Acid Solutions

[0531] Furoic acid or 2-TH acid can be employed topically, for vaginalinstallation, for inhalation or as a mouthwash as a 0.001% to 99.9%,preferably 1% to 50%, most preferably 5% to 20% aqueous solution. Thepreparation can be used in any pharmaceutically acceptable mannerincluding topically, orally, on the mucosa and so forth. It will benoted that furoic acid or 2-TH acid derivatives can be used in place ofthe 2-TH acid, if desired. For inhalation purposes, the solution may beatomized with the use of an appropriate device.

[0532] As stated above, it is likely that furoic acid or 2-TH acid willinterfere with the replication of the retroviruses by chelating zinc andiron and preventing the activity of certain zinc and iron containingproteins. Therefore, a suitable preparation of a chelating material, forexample, 2-TH acid or derivative may be used for vaginal application toprevent infection with any virus containing zinc finger proteins as anessential component of the viral replicating machinery. Such virusesinclude, but are not limited to the families described elsewhere in thisapplication and shown in Table 2. As explained above, the 2-TH acid andsubstituted derivatives thereof are used to attack proteins having zincfinger segments, which are essential for packaging RNA in the viralparticles.

[0533] The preparation may be produced by incorporating approximately 5%to 20% 2-TH acid in a suitable base and instilling the ointmentvaginally before coitus. Such preparations may be used prophylacticallyto prevent infection with these viruses.

[0534] Furthermore, the preparations may be used vaginally to treat theuterine cervix infected with papilloma virus.

EXAMPLE 3 Ocular Preparation

[0535] A preparation of 2-TH acid or a derivative thereof can beprepared for the treatment of ocular herpes or other viral infections ofthe eyes. The topical or intraocular ophthalmological preparationincludes approximately 0.01% to approximately 5% 2-TH acid or one of itssubstituted derivatives in an appropriate, ion-free vehicle, such asmethylcellulose.

EXAMPLE 4 Acne Formulation

[0536] A preparation useful in the treatment and control of acnecomprises approximately 5% to 20% of furoic acid or 2-TH acid, byweight, in a suitable topical lotion. The acne preparation can includeapproximately 1% to approximately 99% of furoic acid or 2-TH acid,derivative or analog thereof. A preferred range is approximately 5% toapproximately 20%. The lotion is applied to the skin two or three timesdaily.

EXAMPLE 5 Intranasal and Inhalation Formulations

[0537] A product suitable for intranasal administration for treatment ofupper respiratory diseases includes approximately 10 mM furoic acid or 5mM 2-TH acid in a suitable isotonic vehicle. The intranasal solution canbe used in a range between 0.01 mM to 10 mM, preferably 0.1 mM up to 20mM furoic acid or 2-TH acid or greater.

[0538] Likewise, a solution for pulmonary inhalation is prepared byadding furoic acid or 2-TH acid to normal saline for nebulization, theresulting solution being in a range of 0.001% to 50% furoic acid or 2-THacid, derivative or analog in saline or sterile distilled water fornebulization.

EXAMPLE 6 Systemic Administration

[0539] A systemic preparation of 2-TH acid, its derivatives or analogscontaining approximately 1% to 100% active ingredient may beadministered orally, intravenously or by any acceptable route for thetreatment of cancer, systemic infections, inflammation orneurodegenerative diseases. For example, 2-TH acid prepared in 00gelatin capsules at 500 mg per capsule may be used to effectivelycontrol metastatic cancer and the associated inflammation. Likewise, aninjectable form may be prepared.

[0540] As set out above, the safe and effective daily systemic dose mayrange for 250 mg to 10 grams for a 70 Kg subject, with the preferredrange being 250 mg to 5 grams, and the most preferred dose being 250 mgto 1000 mg.

We claim:
 1. A method of treating diseases caused by viruses, pathogenicprokaryotic organisms and pathogenic eukaryotic cells, comprising thesystemic administration of an effective amount of a compound having theformula

Wherein R1, which can be singly or multiply substituted in any positionof the thiophene ring not already substituted by R2, is selected fromthe group consisting of hydrogen, alkyl and substituted alkyl whereinthe alkyl portion is from 1 to 21 carbon atoms, inclusive, and isomericforms thereof, cycloalkyls and substituted cycloalkyl, substitutedoxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl,—(CH₂)_(n)—OH, —(CH₂)_(n)—NR3R4, and isomeric forms thereof, wherein nis an integer of from 1 to 21, inclusive, R3 and R4 are H or alkyl offrom 1 to 21 carbon atoms, inclusive, and isomeric forms thereof;wherein R2, which can be singly or multiply substituted in any positionof the thiophene ring not already substituted by R1, is

and X is the amino function of a compound selected from the groupconsisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof, to a mammalhosting a pathogenic virus, prokaryotic pathogenic organism, oreukaryotic pathogenic cell.
 2. A method of treating diseases caused byviruses, pathogenic prokaryotic and eukaryotic pathogenic cells,comprising the systemic administration of an effective amount of acompound having the formula

Wherein A, B, and D are selected from the group consisting of nitrogen,oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim 1, and canbe attached to any ring carbon or nitrogen atom, the R1 can be multiplyattached to any ring carbon atom; the R1 can be 2-carboxylic,2-hydrazine, 2-hydrazone, and 2-thiosemicarbazone; and thepharmaceutically acceptable acid-addition salts thereof to a mammalhosting a pathogenic virus, prokaryotic pathogenic organism, oreukaryotic pathogenic cell.
 3. A method for inactivating ametalloprotein or metalloenzyme of a pathogenic virus, pathogenicprokaryotic organisms or pathogenic eukaryotic cells, wherein the saidprotein comprises an amino acid sequence structure which chelates a zincion or a transition metal ion, said method comprising the step ofcontacting intravirally or intracellularlly the said zinc ion ortransition metal ion bound to the chelating protein structure, with achelating compound which dissociates the metal ion protein complexselected from the group consisting of the following compounds:
 1. Furoicacid having the formula shown in FIG. 1;
 2. Thiophenecaboxylic acidhaving the formula shown in FIG. 1;
 3. Halogenated furoic acid asdefined in claim 1;
 4. Halogenated 2-thiophenecarboxylic acid as definedin claim 1;
 5. Hydrazines of furoic acid or 2-thiophenecarboxylic acidhaving the formula —C—NH—NH—R, where —C is attached to position 2 of thethiophene ring;
 6. Hydrazones of furoic acid or 2-thiophenecarboxylicacid having the formula —CH═N—NH—C═O—, where —C is attached to position2 of the thiophene ring;
 7. Thiosemicarbazones having the formula—CH═N—NH—C═S—, where C is in position 2 of the thiophene ring; 8.Halogenated furoic acid, halogenated 2-thiophenecarboxylic acid, andderivatives thereof where the halogen is selected from the groupconsisting of F,I,Br, and Cl; The compounds of 1 to 8 in which one ormore of the ring residues in positions 3 or 4 have been replaced by afatty acid side chain of 2 to 21 carbons. The compounds of 1 to 8coordinately complexed to cupric ions or to ferric ions, or to toxicmetals such as platinum.
 4. A method for inactivating a metalloproteinor metalloenzyme of a pathogenic virus, pathogenic prokaryotic organismsor pathogenic eukaryotic cells, wherein the said protein comprises anamino acid sequence structure which chelates a zinc ion or a transitionmetal ion, said method comprising the step of contacting intravirally orintracellularly the said zinc ion or transition metal ion bound to thechelating protein structure with a chelating compound which dissociatesthe metal ion protein complex selected from the group consisting of thefollowing compounds: 2-furoic acid; 2-furoic acid hydrazide;Tetrahydro-2-furoic acid; 3,5-dibromo-2-furoic acid;3,4,5-tribromo-2-furoic acid; 2,5-dimethoxytetrahydro-2-furoic acidhydrazine; 5-[2-Chloro-5-(trifluoromethyl)phenyl]-2-furoic acid;5-[3-(trifluoromethyl)phenyl]-2-furoic acid; 5-(2-Nitrophenyl)-2-furoicacid; 5-(3-Nitrophenyl)-2-furoic acid;5-(4-Chloro-2-nitrophenyl)-2-furoic acid; 5-(4-Chlorophenyl)-2-furoicacid; 5-(4-Methyl-2-nitrophenyl)-2-furoic acid;5-(4-Nitrophenyl)-2-furoic acid; 5-Bromo-2-furoic acid;5-Chloro-2-furoic acid; 5-Nitro-2-furoic acid; 5-Nitrofuran-2-carboxylicacid; 2-thiophenecarboxylic acid; 3,4,5-trichloro-2-thiophenecarboxylicacid; 2-thiophenecarboxylic acid hydrazide; 2-thiophenecarboxylic acidhydrazone; 2-thiophenecarboxylic acid thiosemicarbazone;3-methyl-2-thiophenecarboxylic acid; 5-bromo-2-thiophenecarboxylic acidhydrazone; 5-methyl-2-thiophenecarboxylic acid;5-chloro-2-thiophenecarboxylic acid; and 5-chloro-2-thiophenecarboxylicacid hydrazone;
 5. The method according to claim 1, wherein saidcompound is selected from the group consisting of compounds defined inclaims 1,2,3 and
 4. 6. The method according to claim 1 and 2, whereinsaid compounds are selected from the group consisting of compounds inwhich the hydrogen in positions 3 or 4 have been replaced by a fattyacid side chain of 2 to 21 carbons.
 7. The method according to claim 1and 2, wherein the compound is selected from the group consisting ofhydrazones having the formula —C═NH—NH—C═O—, where —C is attached toposition
 2. 8. The method according to claim 1, wherein the compound isFuroic acid.
 9. The method as defined in claim 1, wherein the compoundis 2-Thiopehencarboxylic acid.
 10. The method as defined in claim 1 and2, wherein the compound is selected from the group consisting ofthiosemicarbazones having the formula C═NH—NH—C═S—, where —C is attachedto position
 2. 11. The method as defined in claim 1, wherein thecompound is 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazide.12. The method as defined in claim 1, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone.
 13. Themethod as defined in claim 1, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 14.The method as defined in claim 1 and 2, in which the compound has atleast on functional group capable of binding to the catalytic orstructural transition metal ion chelated to the target protein, inaccordance to claim 3, have at least one functional group which canH-bond with the enzyme or metalloprotein backbone, and have one or moreside chains capable of favorable London interactions with the enzymeactive site or protein structural site.
 15. A method of treating adisease selected from the group consisting of virus infections,bacterial infections, fungal infections, parasitic diseases,degenerative diseases, inflammatory diseases, proliferative diseases, orcancer, wherein the disease is mediated by a metalloprotein having atransition metal ion-protein complex, the method comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2 to inactivate the metal-ion protein complex, wherein R2is carboxylic acid, R1 is in the 3-position or 4-position and is analkyl of from 1 to 21 carbon atoms, and isomeric forms thereof; whereinR2 is in the 3 or 5 position and is an halogen, and the pharmaceuticallyacceptable acid-addition salts thereof, to a mammal hosting a virus,bacteria, fungus, parasites, degenerative diseases, proliferativediseases or cancer.
 16. A method of preventing and treating a viraldisease selected from the group consisting of viral infections in whichviral metalloproteins containing transition metal ions are essential tothe initiation and progression of the disease, comprising the topical orsystemic administration of a therapeutically effective dose of a metalchelating agent to chelate a transition metal ion essential to themetalloprotein, wherein the metal chelating agent is2-thiophenecarboxylic acid or a derivative thereof.
 17. The method ofclaim 16, wherein said compound is selected from the group consisting ofcompounds defined in claim 1,2,3 and
 4. 18. A method for preventing andtreating a systemic viral disease, said disease being smallpox in asubject wherein proteins essential to the initiation and progression ofsmallpox include transition metal ion containing metalloproteins,comprising the topical or systemic administration of a therapeuticallyeffective dose of a metal chelating agent to inactivate a transitionmetal ion essential to the smallpox virus metalloproteins, wherein themetal chelating agent is 2-thiophenecarboxylic acid or a derivativethereof.
 19. The method of claim 18, wherein said compound is selectedfrom the group consisting of compounds defined in claim 1,2,3 and
 4. 20.The method of claim 18, comprising the systemic administration of aneffective amount of a compound, as defined in claims 1 and 2, wherein R1is in the 4-position and is a fatty acid of from 1 to 21 carbon atoms,inclusive, and isomeric forms thereof; wherein R2 is in the 3 or 5position and is an halogen; and the pharmaceutically acceptableacid-addition salts thereof, to a mammal hosting a pathogenic organismor diseased cells.
 21. The method of claim 18, comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-bultyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 22.The method of claim 18, wherein said viral metalloprotein is present inan intact virus.
 23. The method of claim 18, wherein said protein isribonucleotide reductase of smallpox virus.
 24. The method of claim 18,wherein said viral protein is a smallpox nucleocapsid protein.
 25. Themethod of claim 1 and 2, wherein said protein is nucleoprotein np7 of anAIDS virus.
 26. The method of claim 1 and 2, wherein said proteins arezinc finger proteins E6 and E7 of HPV virus.
 27. The method of claim 1,wherein said protein is the MP protein of the influenza virus.
 28. Themethod of claim 18, wherein said virus infected cells is selected fromthe group consisting of viruses carrying metalloproteins in their viralstructures.
 29. A method for preventing and treating a bacterialdisease, said disease being pulmonary Anthrax in a subject whereinproteins essential to the initiation and progression of the diseaseinclude Lethal Factor metalloproteinase of B. Anthraxis which containszinc ions for activity, comprising the topical or systemicadministration of a therapeutically effective dose of a metal chelatingagent to inactivate the zinc ions which are essential to the functioningof the B. Anthraxis Lethal Factor metalloproteinase, wherein the metalchelating agent is 2-thiophenecarboxylic acid or a derivative thereof.30. The method of claim 29, wherein said compound is selected from thegroup consisting of compounds defined in claim 1,2,3 and
 4. 31. A methodof treating a bacterial disease, said disease being pulmonary Anthraxcomprising the systemic administration of an effective amount of acompound, as defined in claims 1 and 2, wherein R1 is in the 4-positionand is a fatty acid of from 1 to 21 carbon atoms, inclusive, andisomeric forms thereof; wherein R2 is in the 3 or 5 position and is anhalogen; and the pharmaceutically acceptable acid-addition saltsthereof, to a mammal hosting a pathogenic B. Anthraxis spore orbacteria.
 32. A method of treating bacterial diseases, comprising thesystemic administration of an effective amount of a compound, as definedin claims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 33.The method of claim 29, wherein said protein is the Lethal Factormetalloproteinase of B. Anthracis.
 34. A method for preventing andtreating a fungal disease, said disease being pulmonary Aspergilosis ina subject wherein proteins essential to the initiation and progressionof the disease include fungal metalloproteinases containing transitionmetal ion for activity, comprising the topical or systemicadministration of a therapeutically effective dose of a metal chelatingagent to inactivate a transition metal ion essential to themetalloprotein complex, wherein the metal chelating agent is2-thiophenecarboxylic acid or a derivative thereof.
 35. The method ofclaim 34, wherein said compound is selected from the group consisting ofcompounds defined in claim 1,2,3 and
 4. 36. A method of treating afungal disease such as pulmonary Aspergillosis comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2, wherein R1 is in the 4-position and is a fatty acid offrom 1 to 21 carbon atoms, inclusive, and isomeric forms thereof;wherein R2 is in the 3 or 5 position and is an halogen; and thepharmaceutically acceptable acid-addition salts thereof, to a mammalhosting a pathogenic organism or diseased cells.
 37. A method oftreating fungal diseases, according to claim 34, comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 38.The method of claim 34, wherein said fungal protein inactivated by thecompounds of claims 1,2,3 is the Toxic Factor of the A. Aspergillus. 39.A method for preventing and treating a parasitic disease, said diseasesbeing Trypanosomiais, Toxoplasmosis or Malaria in a subject whereinproteins essential to the initiation and progression of the diseaseinclude metalloproteinases containing transition metal ion for activity,comprising the topical or systemic administration of a therapeuticallyeffective dose of a metal chelating agent to inactivate a transitionmetal ion essential to the metalloprotein, wherein the metal chelatingagent is 2-Thiophenecarboxylic acid or a derivative thereof.
 40. Themethod of claim 39, wherein said compound is selected from the groupconsisting of compounds defined in claim 1,2,3 and
 4. 41. A method ofpreventing and treating parasitic diseases, said disease beingTrypanosomiasis, Toxoplasmosis, and Malaria, comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2, wherein R1 is in the 4-position and is a fatty acid offrom 1 to 21 carbon atoms, inclusive, and isomeric forms thereof;wherein R2 is in the 3 or 5 position and is an halogen; and thepharmaceutically acceptable acid-addition salts thereof, to a mammalhosting a pathogenic organism or diseased cells.
 42. A method ofpreventing and treating parasitic diseases, said disease beingTrypansomiasis, Toxoplasmosis, or Malarial diseases, comprising thesystemic administration of an effective amount of a compound, as definedin claims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 43.The method of claim 41, wherein said protein is metallopanstimulin/S27ribosomal protein, a zinc-finger protein of Trypanosoma, Toxoplasma orMalaria parasites.
 44. A method of preventing and treating cancer andmetastatic disease, said cancers being pulmonary, prostate, brain,colon, liver and breast, in a subject wherein proteins essential to theinitiation and progression of the disease include transition metal ioncontaining protein complexes, comprising the topical or systemicadministration of a therapeutically effective dose of a metal chelatingagent to inactivate a transition metal ion essential to themetalloprotein, wherein the metal chelating agent is2-Thiophenecarboxylic acid or a derivative thereof.
 45. The method ofclaim 44, wherein said compound is selected from the group consisting ofcompounds defined in claim 1,2,3 and
 4. 46. A method of treating cancerand metastatic disease, said cancers being pulmonary, prostate, brain,colon, liver and breast cancers, comprising the systemic administrationof an effective amount of a compound, as defined in claims 1 and 2,wherein R1 is in the 4-position and is a fatty acid of from 1 to 21carbon atoms, inclusive, and isomeric forms thereof; wherein R2 is inthe 3 or 5 position and is an halogen; and the pharmaceuticallyacceptable acid-addition salts thereof, to a mammal hosting a thecarcinogenic cells.
 47. A method of treating cancer and metastaticdiseases according to claim 44, comprising the systemic administrationof an effective amount of a compound, as defined in claims 1 and 2,wherein the compound is 4-butyl-3-5-dichloro-2-thiophenecarboxylic acidhydrazine, is 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazoneor 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone.48. The method of claim 44, wherein said protein is theMetallopanstimulin/S27 ribosomal protein of cancer cells or metastaticcells.
 49. The method of claim 44, wherein said protein isribonucleotide reductase of cancer cells or metastatic cells.
 50. Themethod of claim 44, wherein said protein is a carcinogenic zinc fingerprotein.
 51. The method of claim 44, wherein said protein is a cancercell or metastatic cell matrix metalloprotease.
 52. A method ofpreventing and treating neurodegenerative diseases, said disease beingAlzheimer's disease, in a subject wherein proteins essential to theinitiation and progression of the disease include metalloproteinscontaining transition metal ion for activity, comprising the topical orsystemic administration of a therapeutically effective dose of a metalchelating agent to inactivate a transition metal ion essential to theactivity of the metalloprotein, wherein the metal chelating agent is2-thiophenecarboxylic acid or a derivative thereof.
 53. The method ofclaim 52, wherein said compound is selected from the group consisting ofcompounds defined in claim 1,2,3 and
 4. 54. A method of treatingneurodegenerative diseases, said disease being Alzheimer's disease,comprising the systemic administration of an effective amount of acompound, as defined in claims 1 and 2, wherein R1 is in the 4-positionand is a fatty acid of from 1 to 21 carbon atoms, inclusive, andisomeric forms thereof; wherein R2 is in the 3 or 5 position and is anhalogen; and the pharmaceutically acceptable acid-addition saltsthereof, to a mammal hosting the pathogenic metalloprotein in theneuronal cells.
 55. A method of treating Alzheimer's disease accordingto claim 52, comprising the systemic administration of an effectiveamount of a compound, as defined in claims 1 and 2, wherein the compoundis 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 56.The method of claim 52, wherein said protein is neuronal amyloid.
 57. Amethod of preventing and treating diseases mediated byneovascularization said disease being cancer or metastatic disease in asubject wherein proteins essential to the initiation and progression ofthe disease include metalloproteinases containing transition metal ionfor activity, comprising the topical or systemic administration of atherapeutically effective dose of a metal chelating agent to inactivatea transition metal ion essential to the activity of the metalloprotein,wherein the metal chelating agent is thiophenecarboxylic acid or aderivative thereof.
 58. The method of claim 57, wherein said compound isselected from the group consisting of compounds defined in claim 1,2,3and
 4. 59. A method of treating pathogenic neovascularization, saidneovascularization occurring in cancer and metastatic disease,comprising the systemic administration of an effective amount of acompound, as defined in claims 1 and 2, wherein R1 is in the 4-positionand is a fatty acid of from 1 to 21 carbon atoms, inclusive, andisomeric forms thereof; wherein R2 is in the 3 or 5 position and is anhalogen; and the pharmaceutically acceptable acid-addition saltsthereof, to a mammal hosting the pathogenic cells mediatingneovascularization.
 60. A method of treating diseases mediated byneovascularization according to claim 57, comprising the systemicadministration of an effective amount of a compound, as defined inclaims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 61.The method of claim 57, wherein said neovascularization-mediatingproteins are macrophage and endothelial cells metalloproteinases.
 62. Amethod of treating organ transplant rejection, said disease being lungtransplant rejection in a subject wherein proteins essential to theinitiation and progression of the rejection include metalloproteinasescontaining transition metal ion for activity, comprising the topical orsystemic administration of a therapeutically effective dose of a metalchelating agent to inactivate a transition metal ion essential toactivity of the metalloproteinase, wherein the metal chelating agent is2-Thiophenecarboxylic acid or a derivative thereof.
 63. The method ofclaim 62, wherein said compound is selected from the group consisting ofcompounds defined in claim 1,2,3 or
 4. 64. A method of treating organtransplant rejection disease, said rejection disease being pulmonarytransplant rejection, comprising the systemic administration of aneffective amount of a compound, as defined in claims 1 and 2, wherein R1is in the 4-position and is a fatty acid of from 1 to 21 carbon atoms,inclusive, and isomeric forms thereof; wherein R2 is in the 3 or 5position and is an halogen; and the pharmaceutically acceptableacid-addition salts thereof, to a mammal hosting a pathogenic organismor diseased cells.
 65. A method of treating transplanted organ rejectiondiseases according to claim 62, comprising the systemic administrationof an effective amount of a compound, as defined in claims 1 and 2,wherein the compound is 4-butyl-3-5-dichloro-2-thiophenecarboxylic acidhydrazine, is 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazoneor 4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone.66. The method of claim 62, wherein said protein is a macrophagemetalloproteinase.
 67. A method of treating inflammatory diseases, saiddisease being rheumatoid arthritis in a subject wherein proteinsessential to the initiation and progression of the disease includemetalloprotein containing transition metal ion for activity, comprisingthe topical or systemic administration of a therapeutically effectivedose of a metal chelating agent to inactivate a transition metal ionessential to the activity of the metalloproteins, wherein the metalchelating agent is 2-Thiphenecarboxylic acid or a derivative thereof.68. The method of claim 67, wherein said compound is selected from thegroup consisting of compounds defined in claim 1,2,3 or
 4. 69. A methodof treating inflammatory disease, said disease being rheumatoidarthritis, comprising the systemic administration of an effective amountof a compound, as defined in claims 1 and 2, wherein R1 is in the4-position and is a fatty acid of from 1 to 21 carbon atoms, inclusive,and isomeric forms thereof; wherein R2 is in the 3 or 5 position and isan halogen; and the pharmaceutically acceptable acid-addition saltsthereof, to a mammal hosting the pathogenic cells.
 70. A method oftreating inflammatory diseases said disease being rheumatoid arthritis,comprising the systemic administration of an effective amount of acompound, as defined in claims 1 and 2, wherein the compound is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazine, is4-butyl-3-5-dichloro-2-thiophenecarboxylic acid hydrazone or4-butyl-3-5-dichloro-2-thiophenecarboxylic acid thiosemicarbazone. 71.The method of claim 70, wherein said protein is DnaJ inflammatorymetalloprotein of macrophages.
 72. The method of claim 1 and 2 whereinthe method of systemic administration of the metal chelating agent isselected from the group comprising topical administration, transdermaladministration, oral administration, parenteral administration, rectaladministration, inhalation administration, intraocular, intravaginal,intraperitoneal administration, and transdermal administration.
 73. Themethod of claims 1 and 2 wherein from about 0.5 to about 500 mg ofcompound per kg of host body weight is systemically administered dailyin association with a pharmaceutical carrier.
 74. The method of claims 1and 2 wherein from about 0.5 to about 500 mg of compound per kg of hostbody weight is parenterally administered daily in association with asterile pharmaceutical carrier.
 75. The method of claim 1 and 2 whereinfrom about 0.5 to about 500 mg of compound per kg of host body weight isorally administered daily in association with a pharmaceutical carrier.76. The method of claim 1 and 2 wherein from about 0.5 to about 500 mgof compound per kg of host body weight is transdermically administereddaily in association with a sterile pharmaceutical carrier.
 77. A methodof preventing and treating radioactive or non-radioactive heavy metalpoisoning disease, said metal being uranium, molybdenum, rhenium,titanium, vanadium, germanium, or tungsten, in a subject whereinproteins essential to the normal functioning of the cells includemetalloproteins intoxicated by said heavy metals, comprising the topicalor systemic administration of a therapeutically effective dose of ametal chelating agent having the composition of claim 1 and 2, tochelate, inactivate, and elute, the toxic heavy metal bound to themetalloprotein, wherein the metal chelating agent is2-thiophenecarboxylic acid or a derivative thereof.
 78. The method ofclaim 77, wherein said compound is selected from the group consisting ofcompounds defined in claims 1,2,3 and
 4. 79. The method of claim 77,wherein said protein is a heavy metal binding protein.
 80. The method ofclaim 77, wherein from about 0.5 to about 500 mg of compound per kg ofhost body weight is parenterally administered daily to detoxify theheavy metal ions from the mammalian hosting the intoxicated cells.
 81. Acompound having the formula:

Wherein R1, which can be singly or multiply substituted in any positionof the thiophene ring not already substituted by R2, is selected fromthe group consisting of hydrogen, alkyl and substituted alkyl whereinthe alkyl portion is from 1 to 21 carbon atoms, inclusive, and isomericforms thereof, cycloalkyls and substituted cycloalkyl, substitutedoxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl,—(CH₂)_(n)—OH, —-(CH₂)—NR3R4, and isomeric forms thereof, wherein n isan integer of from 1 to 21, inclusive, R3 and R4 are H or alkyl of from1 to 21 carbon atoms, inclusive, and isomeric forms thereof; wherein R2,which can be singly or multiply substituted in any position of thethiophene ring not already substituted by R1, is

and X is the amino function of a compound selected from the groupconsisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone.
 82. Acompound having the formula:

Wherein A, B, and D are selected from the group consisting of nitrogen,oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim 1, and canbe attached to any ring carbon or nitrogen atom, the R1 can be multiplyattached to any ring carbon atom; the R1 can be 2-carboxylic,2-hydrazine, 2-hydrazone, and 2-thiosemicarbazone.
 83. A compoundaccording to claims 81 and 82, wherein said compounds are selected fromthe group consisting of compounds in which the hydrogen in positions 3or 4 have been replaced by a fatty acid side chain of 2 to 21 carbons.84. A compound according to claims 81 and 82, wherein the compound isselected from the group consisting of hydrazones having the formula—C═N—NH—C═O—, where —C is attached to position
 2. 85. A compoundaccording to claims 81 and 82, wherein the compound is selected from thegroup consisting of thiosemicarbazones having the formula —C═NH—NH—C═S—,where —C is attached to position
 2. 89. A composition comprising atleast two compounds from the following compound groups represented bythe formula:

Wherein R1, which can be singly or multiply substituted in any positionof the thiophene ring not already substituted by R2, is selected fromthe group consisting of hydrogen, alkyl and substituted alkyl whereinthe alkyl portion is from 1 to 21 carbon atoms, inclusive, and isomericforms thereof, cycloalkyls and substituted cycloalkyl, substitutedoxygen, substituted nitrogen, halogen, phenyl, and substituted phenyl,—(CH₂)_(n)—OH, —(CH₂)_(n)—NR3R4, and isomeric forms thereof, wherein nis an integer of from 1 to 21, inclusive, R3 and R4 are H or all of from1 to 21 carbon atoms, inclusive, and isomeric forms thereof, wherein R2,which can be singly or multiply substituted in any position of thethiophene ring not already substituted by R1, is

and X is the amino function of a compound selected from the groupconsisting of 2-hydrazine, 2-hydrazone, or 2-thiosemicarbazone.
 90. Acomposition comprising at least two compounds from the followingcompound groups having the formula:

Wherein A, B, and D are selected from the group consisting of nitrogen,oxygen, sulfur, and CR1R2; R1 and R2 are as defined in claim 1, and canbe attached to any ring carbon or nitrogen atom, the R1 can be multiplyattached to any ring carbon atom; the R1 can be 2-carboxylic,2-hydrazine, 2-hydrazone, and 2-thiosemicarbazone.
 91. A compositioncomprising at least two compounds from the following compound groups: 1.A compound comprising hydrazines of furoic acid having the formula—C—NH—NH—R, where —C is attached to position 2 of the thiophene ring; 2.A compound comprising hydrazones of furoic acid having the formula—CH═N—NH—C═O—, where —C is attached to position 2 of the thiophene ring;3. A compound comprising thiosemicarbazones of furoic acid having theformula —CH═N—NH—C═S—, where C is in position 2 of the thiophene ring;4. A compound comprising halogenated furoic acid and derivatives thereofwhere the halogen is selected from the group consisting of F,I,Br, andCl; The compounds of 1 to 4 coordinately complexed to cupric ions or toferric ions, or to toxic metals such as platinum.
 92. A compositioncomprising at least two compounds from the following compound groups: 1.A compound comprising hydrazines of 2-thiophenecarboxylic acid havingthe formula —C—NH—NH—R, where —C is attached to position 2 of thethiophene ring;
 2. A compound comprising hydrazones of2-thiophenecarboxylic acid having the formula —CH═N—N—H—C═O—, where —Cis attached to position 2 of the thiophene ring;
 3. A compoundcomprising thiosemicarbazones of 2-thiophenecarboxylic acid having theformula —CH═N—NH—C═S—, where C is in position 2 of the thiophene ring;4. A compound comprising halogenated 2-thiophenecarboxylic acid, andderivatives thereof where the halogen is selected from the groupconsisting of F,I,Br, and Cl; The compounds of 1 to 4 coordinatelycomplexed to cupric ions or to ferric ions, or to toxic metals such asplatinum.
 93. A composition comprising at least two compounds from thefollowing compound groups:
 1. A compound comprising hydrazines of furoicacid having the formula —C—NH—NH—R, where —C is attached to position 2of the thiophene ring and in which one or more of the ring residues inpositions 3 or 4 have been replaced by a fatty acid side chain of 2 to21 carbons.
 2. A compound comprising hydrazones of furoic acid havingthe formula —CH═N—NH—C═O—, where —C is attached to position 2 of thethiophene ring; and in which one or more of the ring residues inpositions 3 or 4 have been replaced by a fatty acid side chain of 2 to21 carbons.
 3. A compound comprising thiosemicarbazones of furoic acidhaving the formula —CH═N—NH—C═S—, where C is in position 2 of thethiophene ring; and in which one or more of the ring residues inpositions 3 or 4 have been replaced by a fatty acid side chain of 2 to21 carbons.
 4. A compound comprising halogenated furoic acid andderivatives thereof where the halogen is selected from the groupconsisting of F,I,Br, and Cl; and in which one or more of the ringresidues in positions 3 or 4 have been replaced by a fatty acid sidechain of 2 to 21 carbons. The compounds of 1 to 4 coordinately complexedto cupric ions or to ferric ions, or to toxic metals such as platinum.94. A composition comprising at least two compounds from the followingcompound groups:
 1. A compound comprising hydrazines of2-thiophenecarboxylic acid having the formula —C—NH—NH—R, where —C isattached to position 2 of the thiophene ring and in which one or more ofthe ring residues in positions 3 or 4 have been replaced by a fatty acidside chain of 2 to 21 carbons.
 2. A compound comprising hydrazones of2-thiophenecarboxylic acid having the formula —CH═N—NH—C═O—, where —C isattached to position 2 of the thiophene ring; and in which one or moreof the ring residues in positions 3 or 4 have been replaced by a fattyacid side chain of 2 to 21 carbons.
 3. A compound comprisingthiosemicarbazones of 2-thiophenecarboxylic acid having the formula—CH═N—N—NH—C═S—, where C is in position 2 of the thiophene ring; and inwhich one or more of the ring residues in positions 3 or 4 have beenreplaced by a fatty acid side chain of 2 to 21 carbons.
 4. A compoundcomprising halogenated 2-thiophenecarboxylic acid, and derivativesthereof where the halogen is selected from the group consisting ofF,I,Br, and Cl; and in which one or more of the ring residues inpositions 3 or 4 have been replaced by a fatty acid side chain of 2 to21 carbons. The compounds of 1 to 4 coordinately complexed to cupricions or to ferric ions, or to toxic metals such as platinum.
 95. Thecomposition of claims 89 to 94 in the protonated or non-protonated formsaccording to the pH of the environment.
 96. The composition of claims 89to 95, wherein the buffering agent comprises at least one agent selectedfrom a group consisting of polyvinylpyrrolidone, hydroxypropyl,methylcellulose, lactose, microcrystalline cellulose, calcium hydrogenphosphate, magnesium stearate, sodium lauryl sulfate, sobitol, cellulosederivatives, hydrogenated fats, lecithin, vegetable oil, oily esters,ethyl alcohol, methyl, propyl-p-hydroxybenzoates, and mixtures thereof.97. The composition of claims 89 to 95, wherein the diluent agentcomprises at least one agent selected from a group consisting of water,methanol, ethanol, hydrochloric, sulfuric, phosphoric, thiocyanic,fluosilisic, hexafluoroarsenic, hexafluorophosphoric, acetic, succinic,citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic,camphoric, glutaric, glycolic, benzoic, and like acids and mixturesthereof.